ABSTRACT Inherited retinal diseases (IRDs), defined by dysfunction or progressive loss of photoreceptors, are disorders characterized by elevated heterogeneity, both at the clinical and

genetic levels. Our main goal was to address the genetic landscape of IRD in the largest cohort of Spanish patients reported to date. A retrospective hospital-based cross-sectional study was

carried out on 6089 IRD affected individuals (from 4403 unrelated families), referred for genetic testing from all the Spanish autonomous communities. Clinical, demographic and familiar

data were collected from each patient, including family pedigree, age of appearance of visual symptoms, presence of any systemic findings and geographical origin. Genetic studies were

were _PRPH2_, _ABCA4_ and _RS1_ in autosomal dominant (AD), autosomal recessive (AR) and X-linked (XL) NON-RP cases, respectively; _RHO_, _USH2A_ and _RPGR_ in AD, AR and XL for

non-syndromic RP; and _USH2A_ and _MYO7A_ in syndromic IRD. Pathogenic variants c.3386G > T (p.Arg1129Leu) in _ABCA4_ and c.2276G > T (p.Cys759Phe) in _USH2A_ were the most frequent

variants identified. Our study provides the general landscape for IRD in Spain, reporting the largest cohort ever presented. Our results have important implications for genetic diagnosis,

counselling and new therapeutic strategies to both the Spanish population and other related populations. SIMILAR CONTENT BEING VIEWED BY OTHERS GENETIC SPECTRUM OF RETINAL DYSTROPHIES IN

TUNISIA Article Open access 08 July 2020 NATIONWIDE GENETIC ANALYSIS OF MORE THAN 600 FAMILIES WITH INHERITED EYE DISEASES IN ARGENTINA Article Open access 22 May 2023 CLINICAL AND GENETIC

CHARACTERISTICS OF 18 PATIENTS FROM 13 JAPANESE FAMILIES WITH CRX-ASSOCIATED RETINAL DISORDER: IDENTIFICATION OF GENOTYPE-PHENOTYPE ASSOCIATION Article Open access 12 June 2020 INTRODUCTION

Inherited retinal diseases (IRDs) are one of the most heterogeneous clinical and genetical disorders known among all human medical conditions, characterized by the progressive loss of

photoreceptor cells, resulting in severe visual impairment1. IRDs are classified as rare diseases, and their estimated prevalence is about 1 in 10002–40001. IRDs can be classified according

to different clinical or genetic criteria, based upon the primary retinal cell affected (rods, cones, retinal pigment epithelium (RPE), bipolar cells or ganglion cells), the ophthalmological

findings and/or the affected gene found after the genetic testing. All modes of inheritance can be observed (autosomal dominant (AD), autosomal recessive (AR), X-linked (XL), including rare

non-Mendelian forms such as mitochondrial or digenic inheritance patterns). Age of onset of first symptoms (from early childhood to adulthood), rate of progression, association with

extra-ocular symptoms (non-syndromic versus syndromic forms) or causative gene can also help to subclassify the different phenotypes3. The most prevailing form of IRDs is Retinitis

Pigmentosa (RP [MIM: 268000]), which is estimated to affect approximately 1.5 million people worldwide4. RP begins with the degeneration of rod photoreceptors, resulting in night blindness

and characteristic pigmentary changes in the peripheral retina. This is also considered a rod-cone dystrophy due to the subsequent cone photoreceptor death in later stages. Other forms of

IRD, including cone-dominated diseases, are characterized by photophobia, reduced visual acuity and impaired colour vision (i.e. cone dystrophy (CD), achromatopsia, blue cone

monochromatism), as well as generalized retinal degeneration involving simultaneously both cones and rods such as in rod-cone or cone-rod dystrophies (CRD). The most severe form of

non-syndromic IRDs is Leber congenital amaurosis (LCA) characterized by congenital or early childhood blindness. Other IRD forms are characterized by central vision loss affecting primarily

the macula and are therefore acknowledged as Macular Dystrophies (MD), such as Stargardt disease (STGD1 [MIM: 248200]) and Best Vitelliform Macular Dystrophy (VMD2 [MIM: 153700])1.

Conversely, syndromic IRDs are subclassified according to the type of syndrome. The most prevalent is Usher syndrome (sensorineural hearing loss and RP) which can be further subclassified to

Usher type I (USH1 [MIM: 276900]), type II (USH2 [MIM: 276901]) and type III (USH3 [MIM: 276902]), and other syndromes such as Bardet-Biedl (BBS [MIM: 209900]) or Alström (ALMS [MIM:

203800])3. Since the identification of rhodopsin (_RHO_ [MIM: 180380]_)_ in 1990 as the first gene involved in the development of AD-RP5, 270 genes have been additionally described as

causative of IRDs (RetNet, Retinal Information Network; https://sph.uth.edu/retnet/; accessed on March 2020). Some of these genes have been reported in only few families worldwide;

therefore, their individual contribution to IRD prevalence is relatively small. The most prevalent IRD-causing genes across all populations are _ABCA4_ (MIM: 601691), _RHO_, _USH2A_ (MIM:

608400) and _RPGR_ (MIM: 312610)_,_ which account for high percentages of some of the IRD subtypes, i.e. 70–71% in STGD1/AR-MD/AR-CRD6, 19–25% of AD-RP, 10% of AR-RP, and 70% of XL-RP,

respectively1. In addition, although the majority of the causative variants are private, some are more frequent, especially in Spanish families, such as _USH2A_ (GenBank: NM_206933.3)

c.2299delG (p.Glu767SerfsTer21) and c.2276G > T (p.Cys759Phe)7 or _ABCA4_ (GenBank: NM_000350.3) c.3386G > T (p.Arg1129Leu) and c.5882G > A (p.Gly1961Glu)6. The aim of this study is

to present a comprehensive overview of the largest cohort of IRD patients ever reported worldwide and related to IRD in the Spanish population. The presented data includes the presumed

inheritance pattern for the different phenotypic subtypes, mutational spectrum, prevalence of genes carrying likely pathogenic variants and the recurrence of disease related variants.

RESULTS PREVALENCE OF IRD IN SPAIN The number of cases diagnosed as having IRD in our hospital (until August 2019) was 6089, and the last Spanish population registry accounted for 46,722,980

habitants giving us a minimal prevalence of 1:7673 (confidence interval (CI):1:7485–1:7871). Regional distribution of cases and prevalence can be seen in Fig. 1A,B. Considering a worldwide

IRD prevalence of 1:10002–40001, our cohort would represent 20–53% of the total patients with IRD in Spain as shown in Supplementary Table S1. For non-syndromic IRD, our cohort was grouped

in 2 categories: 1703 affected individuals (1335 families), in cone-dominated phenotypes—hereafter “NON-RP”—and 3561 affected cases from 2447 unrelated families within primarily rod

affection—hereafter “RP”. This resulted in a minimal prevalence of 1:27,436 (CI:1:26,192–1:28,804) and 1:13,121 (CI:12,704–13,566) for NON-RP and RP, respectively. Additionally, syndromic

IRD forms represented the smallest fraction accounting for 13.6% of the patients with 825 affected individuals (621 families), resulting in a minimal prevalence of 1:56,634

(CI:1:53,016–1:60,781). In our cohort, we have a higher proportion of cases from Madrid area (26.7%; 1625/6089). INITIAL CLASSIFICATION OF IRD FAMILIES BY CLINICAL TYPE AND SUSPECTED MODE OF

INHERITANCE PRIOR TO GENETIC TESTING Non-syndromic NON-RP and RP cases were categorized by the mode of inheritance (Fig. 2A-I and A-II). Syndromic IRD were categorized by the specific type

of suspected syndrome (Fig. 2A-III), instead of inheritance type, given that most of them were sporadic (53.6%) or had recessive inheritance (40.2%). The remaining 6.2% corresponded to

dominant (0.5%), X-linked (0.7%), mitochondrial inherited disease (0.2%) or non-classificable cases (4.8%). According to this “a priori” diagnosis based on the clinical and familial history

of the patients, the main inheritance pattern in non-syndromic NON-RP and RP was recessive or sporadic, representing the 68% and 75% of cases, respectively. Autosomal dominant and X-linked

forms accounted for 21% and 8% for NON-RP and 15% and 8% for RP, respectively (Fig. 2A-I). Families with no familiar data were annotated as unclassified. Non-syndromic RP represents the most

common phenotype, representing 55.6% of families in our cohort. In the present cohort, 47% of the syndromic IRD index cases (270/577) suffered from USH2, followed by 17% USH1 (98/577), as

well as other very rare syndromes like some atypical forms of Usher syndrome (3%; 16/577) and ciliopathies such as BBS or ALMS (16%; 90/577). A miscellanea of non-ciliopathic syndromes or

unclassified symptoms were presented in 103 index cases. MOLECULAR STUDIES DIAGNOSTIC YIELD Genetic testing was performed in a total of 3951 index cases with available DNA6,7,8,9,10,11

(89.7% of the total cohort), including 1291 NON-RP, 2083 RP, and 577 syndromic IRD patients as shown in Supplementary Fig. S1. The genetic analysis procedures evolved over time since novel

genetic approaches were implemented in our laboratory. A definite genetic diagnosis was established for 53.2% (2100/3951) of cases. For NON-RP families, 754 out of 1291 (58.4%) obtained a

genetic diagnosis; in RP the molecular cause was identified in 1038 out of 2083 (49.8%). Similarly, 53.4% (308/577) of syndromic IRD families were genetically solved (Fig. 2B). FINAL

INHERITANCE PATTERN AND RECLASSIFICATION OF CHARACTERIZED FAMILIES The identification of the causative gene allowed us to reclassify the inheritance mode in 8.2% (146/1792) of the NON-RP and

RP families, thus establishing the final inheritance type (Supplementary Table S2). A comparison between the “a priori_”_ suspected inheritance based on the pedigree and the final

inheritance suggested by the molecular diagnosis was performed in characterized NON-RP and RP families (Fig. 2C). As expected, most sporadic NON-RP (n = 378) and RP cases (n = 379) were

confirmed as having AR inheritance after the genetic testing. The rest of the S cases were reclassified to AD (n = 43) and XL (n = 36) (Supplementary Table S2). Twenty-four cases (11 NON-RP

and 13 RP) with an initial unknown mode of inheritance were classified as: AD (n = 5), AR (n = 16), and XL (n = 3) after the molecular testing. GENE LANDSCAPE In total, 1549 different

pathogenic and likely pathogenic variants were identified in 142 different genes. These included SNVs (Single Nucleotide Variants) and CNVs (Copy Number Variants). As showed in Figs. 3, 4,

5, there was a wide spectrum of genes implicated in IRD, 121 of them represented in 1% or less of the cohort. The 5 most frequent mutated genes were _ABCA4, USH2A, RS1_ (MIM: 300839)_, CRB1_

(MIM: 604210) and _RHO._ In NON-RP families, _PRPH2_ (MIM: 179605), _ABCA4_ and _RS1_ were the most commonly mutated genes, explaining 42.2%, 82.7% and 93.3% of the AD, AR and XL forms,

respectively as shown in Supplementary Table S3. For non-syndromic RP, 207 AD-RP families were genetically characterized with heterozygous variants identified in one of 23 genes found. The

most frequent mutated genes were _RHO_ (65/207; 31.4%) and _PRPF31_ (MIM: 606419) (34/207; 16.4%). In AR-RP families, the most common gene mutated was _USH2A_ (127/666; 19.1%). However, the

number of other disease genes detected was very high (N = 70) in the 666 families characterized. For XL-RP families, both the number of cases and variety of genes were low (N = 9) as shown

in Supplementary Table S4. For these patients, 41.2% (68/165) of the index cases (all males) carried a hemizygous pathogenic variant in _RPGR_ (44 _RPGR_ORF15_ and 24 in the rest of _RPGR_

regions)_,_ and 32.7% (54/165) in _CHM_ (MIM: 300390). A total of 53 cases out of the total 2100 (2.5%) were clinically reconsidered and reclassified after genetic testing: 12 NON-RP were

reclassified as RP (9/754; 1.2%; characterized with _RHO, FSCN2, PRPF8, AHI1, CNGB1, TRPM1_ and _CHM_) or as syndromic 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_), syndromic IRD (1/1038; 0.1%; _MYO7A_) and other visual diseases, such as

exudative familiar vitreoretinopathy, optic atrophy, albinism and gyrate atrophy (4/1038; 0.4%; _FZD4, OPA1, GPR143_ and _OAT_). Moreover, 4 syndromic cases were reclassified to RP group

(4/308; 1.3%; _RDH12, PDE6A_ and _RPGRIP1_). Among syndromic IRD families, the causative gene was identified in 56 families with a diagnosis of USH1, 145 of USH2, 6 of atypical Usher, and

101 of other syndromes including Bardet-Biedl or Alström syndrome, respectively. In USH1, biallelic variants in _MYO7A_ (MIM: 276903) were identified in 30 out of 56 patients (53.5%).

_USH2A_ defects were the main cause of USH2 in 90% (129/145) of the patients. The group “others” was a clinically heterogeneous group of non-Usher cases with a total of 48 involved genes,

with _BBS1_ (MIM: 209901) being the most frequent one (N = 23), as shown in Supplementary Table S5. In addition to clinical and genetic heterogeneity, the group of “others” included in

syndromic IRD also presented unusual modes of inheritance, such as triallelism. In our cohort, 4 possible triallelic cases have been identified, all of them diagnosed with BBS, 3 patients

carrying biallelic _BBS1_ variants together with one allele in _MKKS_ (MIM: 604896)9,12 and one additional case carrying biallelic _MKKS_ variants and one allele in _BBS5_ (MIM: 603650). The

phenotypic modifier effect of the triallelism could only be stablished in two of the 4 families. One has two affected siblings with different phenotypic severity that correlates with the

presence of the third allele; and in the other both affected showed triallelism and have the same clinical manifestation. Although, it could not be stablished in the rest of the families, as

they were sporadic cases. MOST FREQUENT VARIANTS Our findings reflect the high allelic heterogeneity in IRD. We identified 458 different disease-causing variants in 45 genes in cases “a

priori” classified as NON-RP, as well as 836 in 94 genes in the “a priori” RP cases and 295 in 55 genes in the “a priori” syndromic IRD. The most common pathogenic variant detected in our

NON-RP cohort was the previously known missense change _ABCA4_ c.3386G > T (p.Arg1129Leu) (180 mutated alleles of 3,618; 5% of the total pathogenic alleles), presented in 21.5% (162/754)

of the characterized families, in homozygous or compound heterozygous state in 18 and 144 families, respectively (Supplementary Table S6). Among the RP families, the most prevalent

pathogenic variant was the missense change _USH2A_ c.2276G > T (p.Cys759Phe), identified in 106 alleles in 8.4% of the solved families (87/1038); in 19 cases in homozygous and in 68 cases

in compound heterozygous state. In addition, there were 15 other variants present in more than 10 genes (Supplementary Table S6). Some of the mutated genes overlap in NON-RP (Fig. 3), RP

(Fig. 4) and syndromic IRD (Fig. 5). DISEASE-CAUSING VARIANT DISTRIBUTION IN SPAIN Analysis of causing variants by the different Spanish regions resulted in a wide variety of disease-causing

variants. Table 1 shows variants detected in more than 5% of characterized families, by Spanish regions. All these variants are depicted in the Supplementary Table S6 of NON-RP and RP most

frequent causing variants, except for the nonsense variant _PRCD_ (OMIM: 610598) (GenBank: NM_001077620.3) c.64C > T (p.Arg22Ter)_,_ that was found in homozygosity in 3 families,

representing 7.2% (6/83) of the identified alleles from Murcia. DISCUSSION This is the first and largest comprehensive study addressing the prevalence and epidemiology of IRD in the Spanish

population. The cohort here described, comprising 6089 cases from 4403 unrelated families, is not based on a national registry of IRD patients, but it is the outcome of a very wide

recruiting effort of a single center over the last 28 years. An increasing number of centers are currently performing clinical and/or genetic diagnosis of IRD in Spain, therefore our cohort

did not reflect all of IRD patients in our country. Hence, to date, no accurate data about the IRD prevalence in the Spanish population is available. In terms of representation of patients

from the different Spanish regions, our cohort reflects a biased recruitment, being enriched with patients from Madrid and the surrounding regions (i.e. Castile and Leon, Castile-La Mancha,

and Extremadura) probably due to the fact that our hospital has been their referral center during most of the time of the study. Other areas like Andalusia, Catalonia, Navarre or the

Valencian Community had different referral centers and genetic testing is performed locally. In spite of these limitations, the large sample size of our cohort and the exhaustive molecular

analysis performed over the years, together with an overall low genetic heterogeneity in the Spanish population13, have allowed a straightforward extrapolation of prevalent genes and/or

variants in IRD. Considering a worldwide prevalence of 1:10002–40001 and an estimated Spanish population of 46.7 million, our cohort would represent 20–53% of the total patients with IRD in

Spain. Despite numerous studies about the characteristics of the different IRDs in Spain, such as NON-RP and RP have been partially published, still no global overview of NON-RP and RP

diseases using a representative cohort has been addressed yet before in our country. Several studies on IRD have been performed globally (Supplementary Table S7) and, in the two last years,

some including big cohorts14,15,16 or meta-analysis17 have been published, reporting more than 125 genes explaining 55–62% of the families using several molecular techniques to achieve

that14,15,16 as it could be also seen in this study. Other studies focused on stablishing the prevalence of IRD in certain regions has been performed in Western countries and in cohorts of

non-syndromic RP, including Western Australia (1:6000)18 or Maine (1:4756)19, as well as in cohorts with general IRD and an estimated prevalence of 1:3454 in Denmark20 or 1:3856 in Norway21.

However, this prevalence has been reported in areas and populations with low rates of consanguinity and could be higher when consanguinity rate increases2, which is not the common scenario

in Spain nowadays. Our study identified AR inheritance as the most common mode of inheritance for non-syndromic IRD, explaining up to 70–75% NON-RP and RP subcohorts (Fig. 2A-I and A-II). By

contrast, only 7% of our NON-RP and RP families are explained by X-linked genes. These results were consistent with previous studies published18,19,20,21,22. Besides, some cases could be

explained by different molecular mechanisms as the pseudodominance, incomplete penetrance or the presence of two variants in an AR gene in AD a priori families8, so extended segregation

analysis within these families are needed. Additional non-Mendelian transmission patterns were only found in exceptionally rare cases with syndromic IRD, including 3 families carrying

variants affecting the mitochondrial DNA and 4 cases with apparent triallelism in BBS-associated genes. Within syndromic IRD group, most of the cases were explained by AR biallelic monogenic

inheritance. Similar to previous published studies from other countries3,23, Usher syndrome was the most prevalent form of syndromic IRD in our cohort, and more specifically, USH2,

representing almost half of the total syndromic IRD families. The overall diagnostic rate of 53.2% obtained here is similar to other studies previously reported (50–70%)24,25,26. Molecular

studies allowed the identification of the genes responsible for the disease and the reclassification of the inheritance type. In our work, 8.2% of the patients were reclassified after the

detection of the disease-causing variants in genes with a specific inheritance pattern. All of those were or have been previously validated. Moreover, in all the characterized sporadic cases

a more accurate genetic classification and counselling could be done8. Additionally, a 2.5% were clinically reclassified after the genetic testing, due to a poor clinical data acquisition

at the origin center. So, identification of the genetic cause of the disease represents a hallmark for the patients, firstly, regarding genetic counselling and the risk of affectation for

other relatives; and secondly, given the possibility of future recruitments for clinical trials targeting specific genes and variants. A total of 142 different genes were identified as the

cause of IRD in our study, but it is important to notice that each subgroup of the cohort (AD, AR and XL NON-RP and RP) has an enrichment of characterized cases in specific genes. For

instance, _PRPH2_ was mutated in more than a third of AD-NON-RP families, followed by _BEST1_ (MIM: 607854). As expected, _ABCA4_ was the most prevalent gene in AR-NON-RP families. Recent

studies in Norway21 and Korea22 also identified this gene as one of the most prevalent mutated genes. A study published by Birtel et al.27 in patients with MD and cone/cone-rod dystrophy

showed a similar distribution of mutated genes, with _ABCA4_, _PRPH2_ and _BEST1_ responsible for 74% of their solved cases. For the XL-NON-RP subcohort, _RS1_ was the most frequently

mutated gene. Non-syndromic RP presented a wider spectrum of causative genes, with _RHO_, _USH2A_ and _RPGR_ (_RPGR_ORF15_ and the rest of _RPGR_ regions) being the most prevalent ones in

AD-RP, AR-RP and XL-RP subcohorts, respectively. Our findings are in line with those published in other studies8,28. For instance, Hartong et al.3, showed as well _MYO7A_, _USH2A_ and _BBS1_

to be the most frequently mutated genes in USH1, USH2 and BBS, respectively. Other studies in different populations highlighted different genes as the most representative in their IRD

cohorts. For example, Eisenberger et al.24 described _RP1_ (MIM: 603937) (11.3%) and _EYS_ (MIM: 612424) (9.4%) as the most frequent genes in German patients with AR-RP, and Kim et al.22

detected that _EYS_ (22%) and _PDE6B_ (MIM: 180072) (17%) are most frequently involved in AR-RP in Korean patients. _EYS_ was also the most prevalent causative gene in the Japanese

population studied by Maeda et al.29, implicated in 21 out of 33 AR-RP patients (63.6%), whereas in our population _EYS_ was mutated in 5.5% of the families with “a priori” AR-RP diagnosis,

being the fourth most frequent gene, after _USH2A_, _CRB1_ and _ABCA4_. However, the order of the causative genes in AR-RP changes after reviewing the clinical data of _ABCA4_ related IRD

patients, since they were mostly reclassified as NON-RP, downgrading _EYS_ as the third most common gene in AR-RP in our population. This result supports an eastward gradient in the

frequency of _EYS_ variants in patients throughout the world and within Europe, being more frequent in Germany than in Spain. The most frequent causing variants detected in our study

appeared, as expected, in _ABCA4_ and _USH2A_, the most prevalent mutated genes in the Spanish population6,7,8,30. _ABCA4_ c.3386G > T (p.Arg1129Leu) is a variant almost exclusively found

in Spanish NON-RP patients6,30, being probably a Spanish founder mutation31,32. However, _USH2A_ c.2276G > T (p.Cys759Phe) is not exclusive from the Spanish population and has been

reported in other populations33. According to the geographical distribution of the variants within the country, no differences between regions were observed. In NON-RP, the two most common

_ABCA4_ variants were also the most represented in regions with variant frequencies above 5%. Meanwhile, in RP we found a higher representation of the most common _USH2A_ variant, which

appeared above 5% of the total alleles in four regions. Finally, two variants appeared to be more frequent in some regions, i.e. _PRCD_ c.64C > T (p.Arg22Ter) in Murcia and _NR2E3_ (MIM:

604485) (GenBank: NM_014249.4) c.932G > A (p.Arg311Gln) in the Canary Islands, where a founder effect could be happening. Our results delineate the genetic background of the Spanish IRD

patients, indicating a wide range of causative genes involved in the disease. Some of the causing variants identified are also frequent in Europe. Some examples include the _ABCA4_ c.5882G 

> A (p.Gly1961Glu), reported with high prevalence in the Italian, German and Spanish populations30,34,35; the c.2276G > T (p.Cys759Phe) as one of the most frequent variants in _USH2A_,

especially in European countries36, _USH2A_ c.2299delG (p.Glu767SerfsTer21), which is possibly an ancestral European pathogenic variant37; _BBS1_ (GenBank: NM_024649.5) c.1169T > G

(p.Met390Arg), identified previously by Mykytyn et al.38 in 22 North American BBS probands with North European ancestry; and _CRB1_ (GenBank: NM_201253.3) c.2843G > A (p.Cys948Tyr),

identified repeatedly in different European countries39,40. Finally, _RHO_ (GenBank: NM_000539.3) c.1040C > T (p.Pro347Leu), described in the Italian and French populations41,42 and also

in non-European cohorts5,43,44. Variants found in individuals from East Mediterranean and Middle-Eastern countries also appeared in our Spanish cohort: _PRCD_ c.64C > T (p.Arg22Ter) found

by Sharon et al.14 in homozygosis in 15 Israeli Muslim Arab families, and by Beheshtian et al.45 in a Persian family; and _FAM161A_ (MIM: 613596) (GenBank: NM_001201543.2) c.1355_1356delCA

(p.Thr452SerfsTer3), which was identified in Jewish families mainly originating from North African countries46. As mentioned above, the variant in _PRCD_ was found with a higher frequency in

the region of Murcia, and this could be due to the settlement of Muslim populations during several centuries during the Middle Ages13. _FAM161A_ does not have a significant specific

geographical distribution in Spain. Remarkably, we identified three pathogenic variants with high frequency in Spain: _ABCA4_ c.3386G > T (p.Arg1129Leu), previously mentioned; _CERKL_

(MIM: 608381) (GenBank: NM_201548.5) c.847C > T (p.Leu283Phe), first described by Tuson et al.47, and _RP1_ c.1625C > G (p.Ser542Ter) previously described originally as a Spanish

founder pathogenic variant. These three variants had been scarcely reported outside the Spanish population. In the case of _RP1_ c.1625C > G (p.Ser542Ter) variant48, because of its

presence in 11 out of 244 unrelated families, we can extrapolate that it may very well account for approximately 4.5% of all AR-RP cases in the Spanish population. Other groups also

identified this variant in Swiss patients26. In conclusion, this study shows the general landscape of the genetic underpinnings of IRD in Spain and will help design clinical and preventive

healthcare approaches to this disorder in our country. MATERIALS AND METHODS COHORT DESCRIPTION A retrospective analysis was performed including all IRD patients from our Spanish registry at

the Fundación Jiménez Díaz University Hospital (FJD, Madrid, Spain) from 1991 until August 2019. This patient registry includes: all patients referred to the Genetic Service at the FJD for

genetic diagnostic testing and/or counselling due to a previous clinical suspicion of IRD, and patients without genetic analysis in our unit but identified in the shared electronic clinical

history of our same-company hospitals using ICD (International Classification of Diseases) terms. The complete cohort contains 6089 IRD affected cases (including index cases and affected

relatives) belonging to 4403 unrelated families as shown in Supplementary Fig. S1. This study was approved by the Ethics Committee of the FJD under approval number 134/2016_FJD and fulfilled

all the tenets of the Declaration of Helsinki and its further reviews. A written informed consent form was obtained from all the patients or their legal guardians. IRD CLASSIFICATION AND

DIAGNOSTIC CRITERIA During this study, different clinical, demographic and familiar data were collected, including (i) family pedigree; (ii) age at onset of visual acuity loss, extent of

visual field loss, night blindness and/or other early symptoms of retinal dystrophy; (iii) presence of any systemic findings suggestive of syndromic forms of IRD; (iv) geographical origin of

the patients. Clinical diagnosis was based on ophthalmic examination, including measurement of best-corrected visual acuity, visual field testing, fundus examination and, if possible,

full-field electroretinography, fundus autofluorescence and spectral domain optical coherence tomography scan. NON-RP and RP include non-syndromic IRD, and their clinical classification was

done according to previously described criteria6,8. NON-RP group include most patients with CD, CRD and achromatopsia, although some of them were included in the RP group due to incomplete

phenotyping at the moment of the diagnosis. Non-syndromic LCA cases were also included in RP. For NON-RP and RP families, an “a priori” inheritance pattern (AD/AR/XL/sporadic (S)) was

established according to previously described criteria1. The subgroup of XL-RP also included choroideremia cases. For cases not extensively described in the first referral, a generic

classification was made as NON-RP or RP. Criteria for syndromic IRD diagnosis were previously described7,9. Information about the geographic origin of all the IRD cases from Spain was

available in 4668. They are distributed throughout the 17 different Spanish communities (Fig. 1A). INHERITANCE RECLASSIFICATION OF IRD CASES After molecular diagnosis, inherited patterns

were reviewed and compared with “a priori” data of each family. Statistical analysis between these data sets to assess the global association in the NON-RP group was made using the Fisher’s

exact test with a p equal to 0.497. Whereas for the RP group, Chi-square test was used with a p below 0.001. Comparisons for each type of inheritance have also been made with the Fisher’s

exact test in the NON-RP Unclassified subgroup and with the Chi-square test in the rest. Fisher’s exact test was used in those cases in which more than 20% of expected values were below than

5, or at least one of the expected frequencies was below 1. Regarding the significance levels chosen, we have a global comparison for which the significance level is the usual threshold of

0.05 and p-value is not corrected, and several post-hoc comparisons for which Bonferroni’s multiple comparisons adjustment is applied, multiplying the p-values by the number of comparisons.

PREVALENCE OF IRD In this study, we performed a retrospective analysis of the largest cohort of patients with IRD from Spain, whom were recruited during a period of 28 years by a single

center, the FJD. The FJD is a center of reference for molecular diagnosis of IRD from all over the country, especially in some specific autonomous communities, like Castile and Leon,

Castile-La Mancha, Extremadura or Madrid. On the other hand, as we take into account other Spanish regions, there are other centers of reference and the prevalence data obtained is

inconclusive. Prevalence was calculated for each clinical type of the disease by dividing the total number of diagnosed IRD cases by the total population in Spain, published by the Spanish

Statistical Office (INE; http://www.ine.es) in January 2019. GENOMIC SCREENING Genomic DNA samples were obtained from the FJD Biobank from a total of 3951 families (89.7%), including 1291

NON-RP, 2083 non-syndromic RP and 577 syndromic IRD families. Molecular studies were performed using different molecular techniques as shown in Supplementary Table S8. According to the

technology available and the knowledge on the genetic determinants of IRD at the time of the diagnosis, a maximum of 291 different genes involved in IRD were processed for the molecular

characterization (Supplementary S1 Appendix). In these studies, index cases were initially screened, analysed following the American College of Medical Genetics and Genomics (ACMG;

https://www.acmg.net/docs/standards_guidelines_for_the_interpretation_of_sequence_variants.pdf) variants classification guidelines. If potentially disease-causing variants were found,

segregation analysis was performed when DNA samples from relatives were available. In the general description of mutated genes and frequent pathogenic variants, only fully molecularly

characterized index cases were considered. Patients with a heterozygote allele in a recessive gene were counted as uncharacterized. The frequency of recurrent IRD causing variants was

established considering not only the total Spanish population, but also the different geographical regions of Spain (Fig. 1A), in order to assess the possibility of identifying any endemic

or founder effects. Pathogenic variants with a prevalence above 5% in a particular region were recorded, and only those with a higher prevalence were considered for further analysis. DATA

AVAILABILITY Part of the NGS data are available in public, open access repositories such as the European Genome-Phenome Archive (EGA; https://www.ebi.ac.uk/ega/home; EGAD00001005746 and

EGAD00001005498), RD-Connect (https://rd-connect.eu/) and the Collaborative Spanish Variant Server (CSVS; http://csvs.babelomics.org/) as aggregated data. The rest of the data are available

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Article  PubMed  Google Scholar  Download references ACKNOWLEDGEMENTS The authors would like to thank Ignacio Mahillo for the statistical support, and all patients who participated in the

study. FUNDING This work was supported by the Instituto de Salud Carlos III (ISCIII) of the Spanish Ministry of Health (FIS; PI16/00425 and PI19/00321), Centro de Investigación Biomédica en

Red Enfermedades Raras (CIBERER, 06/07/0036), IIS-FJD BioBank (PT13/0010/0012), Comunidad de Madrid (CAM, RAREGenomics Project, B2017/BMD-3721), European Regional Development Fund (FEDER),

the Organización Nacional de Ciegos Españoles (ONCE), Fundación Ramón Areces, Fundación Conchita Rábago and the University Chair UAM-IIS-FJD of Genomic Medicine. Irene Perea-Romero is

supported by a PhD fellowship from the predoctoral Program from ISCIII (FI17/00192). Ionut F. Iancu is supported by a grant from the Comunidad de Madrid (CAM, PEJ-2017-AI/BMD7256). Marta del

Pozo-Valero is supported by a PhD grant from the Fundación Conchita Rábago. Berta Almoguera is supported by a Juan Rodes program from ISCIII (JR17/00020). Pablo Minguez is supported by a

Miguel Servet program from ISCIII (CP16/00116). Marta Corton is supported by a Miguel Servet program from ISCIII (CPII17/00006). The funders played no role in study design, data collection,

data analysis, manuscript preparation and/or publication decisions. AUTHOR INFORMATION Author notes * These authors contributed equally: Irene Perea-Romero, Gema Gordo, Ionut F. Iancu, Marta

Corton and Carmen Ayuso. * A list of authors and their affiliations appears at the end of the paper. AUTHORS AND AFFILIATIONS * Department of Genetics, Health Research Institute-Fundación

Jiménez Díaz University Hospital, Universidad Autónoma de Madrid (IIS-FJD, UAM), Madrid, Spain Irene Perea-Romero, Gema Gordo, Ionut F. Iancu, Marta Del Pozo-Valero, Berta Almoguera, Fiona

Blanco-Kelly, Rosario Lopez-Rodriguez, Isabel Lorda-Sanchez, Inmaculada Martin-Merida, Lucia Pérez de Ayala, Rosa Riveiro-Alvarez, Elvira Rodriguez-Pinilla, Saoud Tahsin-Swafiri, Maria J.

Trujillo-Tiebas, Ana Bustamante-Aragones, Rocio Cardero-Merlo, Ruth Fernandez-Sanchez, Jesus Gallego-Merlo, Ines Garcia-Vara, Ascension Gimenez-Pardo, Laura Horcajada-Burgos, Fernando

Infantes-Barbero, Esther Lantero, Miguel A. Lopez-Martinez, Andrea Martinez-Ramas, Lorena Ondo, Marta Rodriguez de Alba, Carolina Sanchez-Jimeno, Camilo Velez-Monsalve, Cristina Villaverde, 

Olga Zurita, Domingo Aguilera-Garcia, Jana Aguirre-Lamban, Ana Arteche, Diego Cantalapiedra, Patricia Fernandez-San Jose, Liliana Galbis-Martinez, Maria Garcia-Hoyos, Carlos Lombardia, Maria

I. Lopez-Molina, Raquel Perez-Carro, Luciana R. J. Da Silva, Carmen Ramos, Rocio Sanchez-Alcudia, Iker Sanchez-Navarro, Sorina D. Tatu, Elena Vallespin, Fermina Lopez-Grondona, Pablo

Minguez, Almudena Avila-Fernandez, Marta Corton & Carmen Ayuso * Center for Biomedical Network Research on Rare Diseases (CIBERER), Instituto de Salud Carlos III, Madrid, Spain Irene

Perea-Romero, Ionut F. Iancu, Marta Del Pozo-Valero, Berta Almoguera, Fiona Blanco-Kelly, Isabel Lorda-Sanchez, Inmaculada Martin-Merida, Rosa Riveiro-Alvarez, Saoud Tahsin-Swafiri, Maria J.

Trujillo-Tiebas, Ana Bustamante-Aragones, Rocio Cardero-Merlo, Jesus Gallego-Merlo, Ascension Gimenez-Pardo, Fernando Infantes-Barbero, Marta Rodriguez de Alba, Carolina Sanchez-Jimeno, 

Cristina Villaverde, Olga Zurita, Elena Aller, Sara Bernal, Gema Garcia-Garcia, Teresa Jaijo, Guillermo Antiñolo, Montserrat Baiget, Jose M. Millan, Maria J. Ballesta-Martinez, Encarna

Guillen-Navarro, Vanesa Lopez-Gonzalez, Feliciano J. Ramos-Fuentes, Lydia Rodriguez-Peña, Jordi Rosell, Blanca Garcia-Sandoval, Pablo Minguez, Almudena Avila-Fernandez, Marta Corton & 

Carmen Ayuso * Department of Ophthalmology, Health Research Institute-Fundación Jiménez Díaz University Hospital, Universidad Autónoma de Madrid (IIS-FJD, UAM), Madrid, Spain Ester Carreño, 

Belen Jimenez-Rolando & Blanca Garcia-Sandoval * Department of Genetics, Health Research Institute-Fundación Jiménez Díaz University Hospital, Universidad Autónoma de Madrid (IIS-FJD,

UAM), Madrid, Spain Elena Aller, Gema Garcia-Garcia, Teresa Jaijo & Jose M. Millan * Center for Biomedical Network Research on Rare Diseases (CIBERER), Instituto de Salud Carlos III,

Madrid, Spain Maria C. Alonso-Cerezo * Medical Genetics Unit, Pediatrics Service, Hospital Clínico Universitario Virgen de la Arrixaca, IMIB-Arrixaca, Murcia, Murcia, Spain Maria J.

Ballesta-Martinez, Encarna Guillen-Navarro, Vanesa Lopez-Gonzalez & Lydia Rodriguez-Peña * CNAG-CRG, Center for Genomic Regulation, The Barcelona Institute of Science and Technology,

Barcelona, Catalonia, Spain Sergi Beltran * Genetics Section, Hospital Universitario Carlos Haya, Málaga, Andalusia, Spain Carmen Benito Lopez * Department of Genetics, Hospital de la Santa

Creu I Sant Pau, Universitat Autònoma de Barcelona, Barcelona, Catalonia, Spain Sara Bernal & Montserrat Baiget * Ophthalmology Service, Hospital Sant Joan de Déu, Barcelona, Catalonia,

Spain Jaume Català-Mora * Department of Medical Genetics, Hospital Universitario Cruces, Bilbao, Basque Country, Spain Claudio Catalli, Blanca Gener, Isabel Llano-Rivas & Maria A.

López-Ariztegui * Clinical Genetics Unit, Hospital Universitario Clínico San Carlos, Madrid, Madrid, Spain Carmen Cotarelo-Perez & Raluca Oancea-Ionescu * Genetics Unit, Hospital de

Mérida, Mérida, Badajoz, Extremadura, Spain Miguel Fernandez-Burriel * Genetics Service, Hospital Universitario Marqués de Valdecilla (HUMV), Santander, Cantabria, Spain Ana Fontalba-Romero

* Clinical Genetics Unit, Pediatrics Service, Hospital Universitario de Badajoz, Badajoz, Extremadura, Spain Enrique Galán-Gómez & Pilar Mendez-Perez * Molecular Genetics Unit, Hospital

de Terrassa, Consorci Sanitari de Terrassa, Terrassa, Catalonia, Spain Maria J. Gamundi, Inmaculada Hernan & Miguel Carballo * Genetics Unit, Hospital Universitario de Basurto,

Osakidetza Basque Health Service, Bilbao, Basque Country, Spain Maria Garcia-Barcina * Clinical Genetics Unit, Complejo Hospitalario Insular-Materno Infantil, Las Palmas de Gran Canaria,

Canary Islands, Spain Loida M. Garcia-Cruz & Alfredo Santana * Department of Genetics, Hospital Universitario de Getafe, Madrid, Madrid, Spain Belen Gil-Fournier * Genetics Unit,

Hospital Son Espases, Palma de Mallorca, Balearic Islands, Spain Nancy Govea & Jordi Rosell * Genetics Deparment, Hospital Universitario Central de Asturias, Oviedo, Asturias, Spain Ines

Hernando Acero * Department of Ophthalmology, Hospital Universitario de Donostia, San Sebastián, Basque Country, Spain Cristina Irigoyen * Division of Neurosciences, Biodonostia Health

Research Institute, San Sebastián, Basque Country, Spain Cristina Irigoyen * Department of Ophthalmology, University of the Basque Country-UPV/EHU, Vizcaya, Basque Country, Spain Cristina

Irigoyen * Clinical Genetics Department, Hospital Universitario Miguel Servet (HUMS), Zaragoza, Aragon, Spain Silvia Izquierdo-Álvarez * Cellular Therapy Service, Blood and Tissue Bank,

Building Dr. Frederic Duran I Jordà, Barcelona, Catalonia, Spain Loreto Martorell * Musculoskeletal Tissue Engineering Group, Vall D’Hebron Research Institute (VHIR), Universitat Autònoma de

Barcelona, Barcelona, Catalonia, Spain Loreto Martorell * Department of Medical Genetics, Complejo Hospitalario de Navarra (CHN), Pamplona, Navarre, Spain Maria Moreno-Igoa * Navarra

Institute for Health Research (IdiSNA), Pamplona, Navarre, Spain Maria Moreno-Igoa * Molecular Genetics Unit, Hospital Sant Joan de Deu, Barcelona, Catalonia, Spain Francesc Palau-Martinez *

Molecular (Epi)Genetic Lab, Bioaraba Health Research Institute, Araba University Hospital, Vitoria-Gasteiz, Alava, Basque Country, Spain Guiomar Perez de Nanclares * Clinical Genetics and

Functional Genomics Unit, Pediatrics Service, Hospital Clinico Universitario “Lozano Blesa”, Facultad de Medicina, Universidad de Zaragoza, Zaragoza, Aragon, Spain Feliciano J. Ramos-Fuentes

* Department of Clinical Analysis, Hospital Universitario General de Valencia, Valencia, Valencian Community, Spain Raquel Rodriguez-Lopez * Genetics Unit, Hospital Materno Infantil Teresa

Herrera, Hospital Universitario de A Coruña, A Coruña, Galicia, Spain Montserrat Rodriguez-Pedreira & Berta Rodriguez-Sanchez * Instituto Investigación Illes Balears (IDISBA), Palma de

Mallorca, Balearic Islands, Spain Jordi Rosell * Department of Ophthalmology, Hospital de la Santa Creu I Sant Pau, Universitat Autònoma de Barcelona, Barcelona, Catalonia, Spain Noemi

Rosello * Department of Genetics, Hospital Universitario Donostia, San Sebastian, Basque Country, Spain Raquel Saez-Villaverde * Genetics Unit, Hospital Universitari Vall D’Hebron,

Barcelona, Catalonia, Spain Irene Valenzuela-Palafoll * Instituto Oftalmológico Fernández-Vega, Oviedo, Asturias, Spain Eva Villota-Deleu * Department of Ophthalmology, Columbia University,

New York, NY, USA Rando Allikmets * Department of Pathology and Cell Biology, Columbia University, New York, NY, USA Rando Allikmets * Medical Genetics, Department of Precision Medicine,

Università degli Studi della Campania ‘Luigi Vanvitelli’, Naples, Italy Sandro Banfi * Telethon Institute of Genetics and Medicine, Pozzuoli, Italy Sandro Banfi * Department of Human

Genetics, Radboud University Medical Center, Nijmegen, The Netherlands Frans P. M. Cremers * Department of Human Genetics and Donders Institute for Brain, Cognition and Behaviour, Radboud

University Medical Center, Nijmegen, The Netherlands Rob W. J. Collin * Center for Medical Genetics Ghent (CMGG), Ghent University and Ghent University Hospital, Ghent, Belgium Elfride De

Baere * Center for Applied Genomics, Children’s Hospital of Philadelphia, Philadelphia, PA, USA Hakon Hakonarson * Division of Human Genetics, Children’s Hospital of Philadelphia,

Philadelphia, PA, USA Hakon Hakonarson * Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA Hakon Hakonarson * Institute for Ophthalmic

Research, Center for Ophthalmology, University of Tübingen, Tübingen, Germany Susanne Kohl * Clinical Research Center, Institute of Molecular and Clinical Ophthalmology Basel (IOB), Basel,

Switzerland Carlo Rivolta * Department of Ophthalmology, University Hospital Basel, Basel, Switzerland Carlo Rivolta * Department of Genetics and Genome Biology, University of Leicester,

Leicester, UK Carlo Rivolta * Department of Ophthalmology, Hadassah Medical Center, Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem, Israel Dror Sharon * Department of

Maternofoetal Medicine, Genetics and Reproduction, Institute of Biomedicine of Seville (IBIS), University Hospital Virgen del Rocío-CSIC-University of Seville, Seville, Spain Guillermo

Antiñolo * Department of Biochemistry, Genetics and Immunology, Facultad de Biología, Universidad de Vigo, Pontevedra, Galicia, Spain Diana Valverde * Research Group on Rare Diseases and

Pediatric Medicine, Instituto de Investigacion Sanitaria Galicia Sur (IISGS), Vigo, Galicia, Spain Diana Valverde * Centro de Investigaciones Biomédicas (CINBIO), Universidad de Vigo, Vigo,

Galicia, Spain Diana Valverde Authors * Irene Perea-Romero View author publications You can also search for this author inPubMed Google Scholar * Gema Gordo View author publications You can

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inPubMed Google Scholar CONSORTIA THE ESRETNET STUDY GROUP * Ana Bustamante-Aragones * , Rocio Cardero-Merlo * , Ruth Fernandez-Sanchez * , Jesus Gallego-Merlo * , Ines Garcia-Vara * , 

Ascension Gimenez-Pardo * , Laura Horcajada-Burgos * , Fernando Infantes-Barbero * , Esther Lantero * , Miguel A. Lopez-Martinez * , Andrea Martinez-Ramas * , Lorena Ondo * , Marta Rodriguez

de Alba * , Carolina Sanchez-Jimeno * , Camilo Velez-Monsalve * , Cristina Villaverde * , Olga Zurita * , Domingo Aguilera-Garcia * , Jana Aguirre-Lamban * , Ana Arteche * , Diego

Cantalapiedra * , Patricia Fernandez-San Jose * , Liliana Galbis-Martinez * , Maria Garcia-Hoyos * , Carlos Lombardia * , Maria I. Lopez-Molina * , Raquel Perez-Carro * , Luciana R. J. Da

Silva * , Carmen Ramos * , Rocio Sanchez-Alcudia * , Iker Sanchez-Navarro * , Sorina D. Tatu * , Elena Vallespin * , Elena Aller * , Sara Bernal * , Maria J. Gamundi * , Gema Garcia-Garcia *

, Inmaculada Hernan * , Teresa Jaijo * , Guillermo Antiñolo * , Montserrat Baiget * , Miguel Carballo * , Jose M. Millan *  & Diana Valverde THE ERDC STUDY GROUP * Rando Allikmets * , 

Sandro Banfi * , Frans P. M. Cremers * , Rob W. J. Collin * , Elfride De Baere * , Hakon Hakonarson * , Susanne Kohl * , Carlo Rivolta *  & Dror Sharon THE ASSOCIATED CLINICAL STUDY

GROUP * Maria C. Alonso-Cerezo * , Maria J. Ballesta-Martinez * , Sergi Beltran * , Carmen Benito Lopez * , Jaume Català-Mora * , Claudio Catalli * , Carmen Cotarelo-Perez * , Miguel

Fernandez-Burriel * , Ana Fontalba-Romero * , Enrique Galán-Gómez * , Maria Garcia-Barcina * , Loida M. Garcia-Cruz * , Blanca Gener * , Belen Gil-Fournier * , Nancy Govea * , Encarna

Guillen-Navarro * , Ines Hernando Acero * , Cristina Irigoyen * , Silvia Izquierdo-Álvarez * , Isabel Llano-Rivas * , Maria A. López-Ariztegui * , Vanesa Lopez-Gonzalez * , Fermina

Lopez-Grondona * , Loreto Martorell * , Pilar Mendez-Perez * , Maria Moreno-Igoa * , Raluca Oancea-Ionescu * , Francesc Palau-Martinez * , Guiomar Perez de Nanclares * , Feliciano J.

Ramos-Fuentes * , Raquel Rodriguez-Lopez * , Montserrat Rodriguez-Pedreira * , Lydia Rodriguez-Peña * , Berta Rodriguez-Sanchez * , Jordi Rosell * , Noemi Rosello * , Raquel Saez-Villaverde

* , Alfredo Santana * , Irene Valenzuela-Palafoll *  & Eva Villota-Deleu CONTRIBUTIONS Conceptualization, I.P.R., G.G., I.F.I., M.P.V., B.A., F.B.K., E.C., B.J.R., I.M.M., R.R.A.,

B.G.S., P.M., A.A.F., M.C. and C.A.; Data curation, I.P.R., G.G., I.F.I., M.P.V., L.O., L.P.A., C.L. and C.A.; Formal analysis, I.P.R., G.G., I.F.I., M.P.V., B.A., F.B.K., A.B.A., R.C.M.,

E.C., R.F.S., J.G.M., I.G.V., A.G.P., L.H.B., F.I.B., B.J.R., E.L., M.A.L.M., R.L.R., I.L.S., I.M.M., A.M.R., L.O., L.P.A., R.R.A., M.R.A., E.R.P., C.S.J., S.T.S., M.J.T.T., C.V.M., C.V.,

O.Z., D.A.G., A.A., R.P.C., I.S.N., R.A., S.B., F.P.M.C., R.W.J.C., E.D.B., H.H., S.K., C.R., D.S., G.A., M.B., M.C., J.M.M., D.V., B.G.S., P.M., A.A.F., M.C. and C.A.; Funding acquisition,

C.A.; Investigation, I.P.R., G.G., I.F.I., M.P.V., B.A., F.B.K., A.B.A., R.C.M., E.C., R.F.S., J.G.M., I.G.V., A.G.P., L.H.B., F.I.B., B.J.R., E.L., M.A.L.M., R.L.R., I.L.S., I.M.M., A.M.R.,

L.O., L.P.A., R.R.A., M.R.A., E.R.P., C.S.J., S.T.S., M.J.T.T., C.V.M., C.V., O.Z., D.A.G., J.A.L., A.A., D.C., P.F.S.J., L.G.M., M.G.H., C.L., M.I.L.M., R.P.C., L.R.J.D.S., C.R., R.S.A.,

I.S.N., S.D.T., E.V., R.A., S.B., F.P.M.C., R.W.J.C., E.D.B., H.H., S.K., C.R., D.S., G.A., M.B., M.C., J.M.M., D.V., B.G.S., P.M., A.A.F., M.C. and C.A.; Methodology, I.P.R., G.G., I.F.I.,

M.P.V., D.A.G., J.A.L., A.A., D.C., P.F.S.J., L.G.M., M.G.H., C.L., M.I.L.M., R.P.C., L.R.J.D.S., C.R., R.S.A., I.S.N., S.D.T., E.V., E.A., M.J.G., G.G.G., I.H., T.J., R.A., S.B., F.P.M.C.,

R.W.J.C., E.D.B., H.H., S.K., C.R., D.S., G.A., M.B., M.C., J.M.M., D.V., B.G.S., P.M., A.A.F., M.C. and C.A.; Project administration, C.A.; Resources, F.B.K., E.C., B.J.R., I.L.S., E.R.P.,

S.T.S., E.A., M.C.A.C., M.J.B.M., S.B., C.B.L., S.B., J.C.M., C.C., C.C.P, M.F.B., A.F.R., E.G.G., M.J.G., M.G.B., L.M.G.C., G.G.G., B.G., B.G.F., N.G., E.G.N., I.H., I.H.A., C.I., S.I.A.,

T.J., I.L.R., M.A.L.A., V.L.G., F.L.G., L.M., P.M.P., M.M.I., R.O.I., F.P.M., G.P.N., F.J.R.F., R.R.L., M.R.P., L.R.P., B.R.S., J.R., N.R., R.S.V., A.S., I.V.P., E.V.D., R.A., S.B.,

F.P.M.C., R.W.J.C., E.D.B., H.H., S.K., C.R., D.S., G.A., M.B., M.C., J.M.M., D.V., B.G.S., P.M. and C.A.; Software, I.F.I., B.A., R.A., S.A., F.P.M.C., R.W.J.C., E.D.B., H.H., S.K., C.R.,

D.S. and P.M.; Supervision, M.C. and C.A.; Validation, I.P.R., G.G., I.F.I., M.P.V., B.A., F.B.K., A.B.A., R.C.M., E.C., R.F.S., J.G.M., I.G.V., A.G.P., L.H.B., F.I.B., B.J.R., E.L.,

M.A.L.M., R.L.R., I.L.S., I.M.M., A.M.R., L.O., L.P.A., R.R.A., M.R.A., E.R.P., C.S.J., S.T.S., M.J.T.T., C.V.M., C.V., O.Z., D.A.G., J.A.L., A.A., D.C., P.F.S.J., L.G.M., M.G.H., C.L.,

M.I.L.M., R.P.C., L.R.J.D.S., C.R., R.S.A., I.S.N., S.D.T., E.V., E.A., M.C.A.C., M.J.B.M., S.B., C.B.L., S.B., J.C.M., C.C., C.C.P., M.F.B., A.F.R., E.G.G., M.J.G., M.G.B., L.M.G.C.,

G.G.G., B.G., B.G.F., N.G., E.G.N., I.H., I.H.A., C.I., S.I.A., T.J., I.L.R., M.A.L.A., V.L.G., F.L.G., L.M., P.M.P., M.M.I., R.O.I., F.P.M., G.P.N., F.J.R.F., R.R.L., M.R.P., L.R.P.,

B.R.S., J.R., N.R., R.S.V., A.S., I.V.P., E.V.D., G.A., M.B., M.C., J.M.M., D.V., B.G.S., P.M., A.A.F., M.C. and C.A.; Visualization, I.P.R., G.G., I.F.I., M.C. and C.A.; Writing—original

draft, I.P.R., G.G., I.F.I., M.P.V., M.G.H., M.C. and C.A.; Writing—review & editing, all authors. CORRESPONDING AUTHORS Correspondence to Marta Corton or Carmen Ayuso. ETHICS

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inherited retinal dystrophies affected cases in Spain and their therapeutic and extended epidemiological implications. _Sci Rep_ 11, 1526 (2021). https://doi.org/10.1038/s41598-021-81093-y

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