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Purpose We aimed to unravel the molecular basis of sporadic retinitis pigmentosa (sRP) in the largest cohort reported to date. Design Case series. Participants A cohort of 877 unrelated Spanish sporadic cases with a clinical diagnosis of retinitis pigmentosa (RP) and negative family history. Methods The cohort was studied by classic genotyping or targeted next-generation sequencing (NGS). Multiplex ligation-dependent probe amplification (MLPA) and array-based comparative genomic hybridization were performed to confirm copy number variations detected by NGS. Quantitative fluorescent polymerase chain reaction was assessed in sRP cases carrying de novo variants to confirm paternity. Main Outcome Measures The study of the sRP cohort showed a high proportion of causal autosomal dominant (AD) and X-linked (XL) variants, most of them being de novo. Results Causative variants were identified in 38% of the patients studied, segregating recessively in 84.5% of the solved cases. Biallelic variants detected in only 6 different autosomal recessive genes explained 50% of the cases characterized. Causal AD and XL variants were found in 7.6% and 7.9% of cases, respectively. Remarkably, 20 de novo variants were confirmed after trio analysis, explaining 6% of the cases. In addition, 17% of the solved sRP cases were reclassified to a different retinopathy phenotype. Conclusions This study highlights the clinical utility of NGS testing for sRP cases, expands the mutational spectrum, and provides accurate prevalence of mutated genes. Our findings evidence the underestimated role of de novo variants in the etiology of RP, emphasizing the importance of segregation analysis as well as comprehensive screening of genes carrying XL and AD variants in sporadic cases. Such in-depth study is essential for accurate family counseling and future enrollment in gene therapy–based treatments.

We aimed to unravel the molecular basis of sporadic retinitis pigmentosa (sRP) in the largest cohort reported to date.Case series.A cohort of 877 unrelated Spanish sporadic cases with a clinical diagnosis of retinitis pigmentosa (RP) and negative family history.The cohort was studied by classic genotyping or targeted next-generation sequencing (NGS). Multiplex ligation-dependent probe amplification (MLPA) and array-based comparative genomic hybridization were performed to confirm copy number variations detected by NGS. Quantitative fluorescent polymerase chain reaction was assessed in sRP cases carrying de novo variants to confirm paternity.The study of the sRP cohort showed a high proportion of causal autosomal dominant (AD) and X-linked (XL) variants, most of them being de novo.Causative variants were identified in 38% of the patients studied, segregating recessively in 84.5% of the solved cases. Biallelic variants detected in only 6 different autosomal recessive genes explained 50% of the cases characterized. Causal AD and XL variants were found in 7.6% and 7.9% of cases, respectively. Remarkably, 20 de novo variants were confirmed after trio analysis, explaining 6% of the cases. In addition, 17% of the solved sRP cases were reclassified to a different retinopathy phenotype.This study highlights the clinical utility of NGS testing for sRP cases, expands the mutational spectrum, and provides accurate prevalence of mutated genes. Our findings evidence the underestimated role of de novo variants in the etiology of RP, emphasizing the importance of segregation analysis as well as comprehensive screening of genes carrying XL and AD variants in sporadic cases. Such in-depth study is essential for accurate family counseling and future enrollment in gene therapy-based treatments.

Introduction Mutations in USH2A cause both isolated Retinitis Pigmentosa (RP) and Usher syndrome (that implies RP and hearing impairment). One of the most frequent variants identified in this gene and among these patients is the p.(Cys759Phe) change. However, the pathogenic role of this allele has been questioned since it was found in homozygosity in two healthy siblings of a Spanish family. To assess the causative role of USH2A p.(Cys759Phe) in autosomal recessive RP (ARRP) and Usher syndrome type II (USH2) and to establish possible genotype-phenotype correlations associated with p.(Cys759Phe), we performed a comprehensive genetic and clinical study in patients suffering from any of the two above-mentioned diseases and carrying at least one p.(Cys759Phe) allele. Materials and methods Diagnosis was set according to previously reported protocols. Genetic analyses were performed by using classical molecular and Next-Generation Sequencing approaches. Probands of 57 unrelated families were molecularly studied and 63 patients belonging to these families were phenotypically evaluated. Results Molecular analysis characterized 100% of the cases, identifying: 11 homozygous patients for USH2A p.(Cys759Phe), 42 compound heterozygous patients (12 of them with another missense USH2A pathogenic variant and 30 with a truncating USH2A variant), and 4 patients carrying the p.(Cys759Phe) allele and a pathogenic variant in another RP gene (PROM1, CNGB1 or RP1). No additional causative variants were identified in symptomatic homozygous patients. Statistical analysis of clinical differences between zygosity states yielded differences (p

Inherited syndromic retinopathies are a highly heterogeneous group of diseases that involve retinal anomalies and systemic manifestations. They include retinal ciliopathies, other well-defined clinical syndromes presenting with retinal alterations and cases of non-specific multisystemic diseases. The heterogeneity of these conditions makes molecular and clinical characterization of patients challenging in daily clinical practice. We explored the capacity of targeted resequencing and copy-number variation analysis to improve diagnosis of a heterogeneous cohort of 47 patients mainly comprising atypical cases that did not clearly fit a specific clinical diagnosis. Thirty-three likely pathogenic variants were identified in 18 genes (ABCC6, ALMS1, BBS1, BBS2, BBS12, CEP41, CEP290, IFT172, IFT27, MKKS, MYO7A, OTX2, PDZD7, PEX1, RPGRIP1, USH2A, VPS13B, and WDPCP). Molecular findings and additional clinical reassessments made it possible to accurately characterize 14 probands (30% of the total). Notably, clinical refinement of complex phenotypes was achieved in 4 cases, including 2 de novo OTX2-related syndromes, a novel phenotypic association for the ciliary CEP41 gene, and the co-existence of biallelic USH2A variants and a Koolen-de-Vries syndrome-related 17q21.31 microdeletion. We demonstrate that combining next-generation sequencing and CNV analysis is a comprehensive and useful approach to unravel the extensive phenotypic and genotypic complexity of inherited syndromic retinopathies, This study was funded by several grants from the Instituto de Salud Carlos III (ISCIII) from the Spanish Ministry of Health, including CIBERER (06/07/0036), FIS - FEDER (European Regional Development Fund) (PI016/00425), IIS-FJD Biobank PT13/0010/0012. In addition, sponsored chair HU-FJD-UAM “Cátedra de Patrocinio Medicina Genómica”, the Spanish National Organization of the Blind (ONCE) and the Spanish Fighting Blindness Foundation (FUNDALUCE) also supported our work. ISN and RSA are sponsored by Sara Borrell Postdoctoral Program (CD13-00085 and CD12-00676) from ISCIII/FEDER. MC is supported by the Miguel Servet Program (CP12/03256) from ISCIII/FEDER. RPC is supported by Fundación Conchita Rábago and LRJS is supported by CAPES Foundation, Ministry of Education of Brazil

Purpose The aim was to determine the prevalence of PRPF31 mutations in a cohort of Spanish autosomal dominant retinitis pigmentosa (adRP) families to deepen knowledge of the pathogenic mechanisms underlying the disease and to assess genotype-phenotype correlations. Methods A cohort of 211 adRP patients was screened for variants in PRPF31 by using a combined strategy comprising next-generation sequencing approaches and copy-number variation (CNV) analysis. Quantitative RT-PCR and CNV analysis of the regulatory MSR1 element were also performed to assess PRPF31 gene expression. Phenotype was assessed by using ophthalmologic examination protocols. Results Fifteen different causative mutations and genomic rearrangements were identified, revealing five novel mutations. Prevalence of PRPF31 mutations, genomic rearrangements, and lack of penetrance were 7.6%, 1.9%, and 66.7%, respectively. Interestingly, we identified a tandem duplication and a partial PRPF31 deletion in different affected individuals from the same family. PRPF31 gene expression was significantly decreased in symptomatic cases carrying either PRPF31 duplication or deletion as compared to controls. The 4 MSR1 allele in cis with the PRPF31 wild-type allele was apparently a protective factor. The mutated phenotype varied from no symptoms to typical retinitis pigmentosa with variable onset and course depending on the kind of mutation, with the duplication case the most severe. Conclusions In view of the high genetic heterogeneity of PRPF31 mutations, the screening must include the entire gene, as well as CNV assays, to detect large rearrangements. This is the first report of a variable phenotype correlation as well as a gross duplication and deletion within the same family.

Objective To identify the genetic causes underlying autosomal recessive retinitis pigmentosa (arRP) and to describe the associated phenotype. Design Case series. Participants Three hundred forty-seven unrelated families affected by arRP and 33 unrelated families affected by retinitis pigmentosa (RP) plus noncongenital and progressive hearing loss, ataxia, or both, respectively. Methods A whole exome sequencing (WES) analysis was performed in 2 families segregating arRP. A mutational screening was performed in 378 additional unrelated families for the exon–intron boundaries of the ABHD12 gene. To establish a genotype–phenotype correlation, individuals who were homozygous or compound heterozygotes of mutations in ABHD12 underwent exhaustive clinical examinations by ophthalmologists, neurologists, and otologists. Main Outcome Measures DNA sequence variants, best-corrected visual acuity, visual field assessments, electroretinogram responses, magnetic resonance imaging, and audiography. Results After a WES analysis, we identified 4 new mutations (p.Arg107Glufs*8, p.Trp159*, p.Arg186Pro, and p.Thr202Ile) in ABHD12 in 2 families (RP-1292 and W08-1833) previously diagnosed with nonsyndromic arRP, which cosegregated with the disease among the family members. Another homozygous mutation (p.His372Gln) was detected in 1 affected individual (RP-1487) from a cohort of 378 unrelated arRP and syndromic RP patients. After exhaustive clinical examinations by neurologists and otologists, the 4 affected members of the RP-1292 had no polyneuropathy or ataxia, and the sensorineural hearing loss and cataract were attributed to age or the normal course of the RP, whereas the affected members of the families W08-1833 and RP-1487 showed clearly symptoms associated with polyneuropathy, hearing loss, cerebellar ataxia, RP, and early-onset cataract (PHARC) syndrome. Conclusions Null mutations in the ABHD12 gene lead to PHARC syndrome, a neurodegenerative disease including polyneuropathy, hearing loss, cerebellar ataxia, RP, and early-onset cataract. Our study allowed us to report 5 new mutations in ABHD12 . This is the first time missense mutations have been described for this gene. Furthermore, these findings are expanding the spectrum of phenotypes associated with ABHD12 mutations ranging from PHARC syndrome to a nonsyndromic form of retinal degeneration.

Retinitis pigmentosa (RP) is a group of inherited progressive retinal dystrophies (RD) characterized by photoreceptor degeneration. RP is highly heterogeneous both clinically and genetically, which complicates the identification of causative genes and mutations. Targeted next-generation sequencing (NGS) has been demonstrated to be an effective strategy for the detection of mutations in RP. In our study, an in-house gene panel comprising 75 known RP genes was used to analyze a cohort of 47 unrelated Spanish families pre-classified as autosomal recessive or isolated RP. Disease-causing mutations were found in 27 out of 47 cases achieving a mutation detection rate of 57.4%. In total, 33 pathogenic mutations were identified, 20 of which were novel mutations (60.6%). Furthermore, not only single nucleotide variations but also copy-number variations, including three large deletions in the USH2A and EYS genes, were identified. Finally seven out of 27 families, displaying mutations in the ABCA4, RP1, RP2 and USH2A genes, could be genetically or clinically reclassified. These results demonstrate the potential of our panel-based NGS strategy in RP diagnosis.

Choroideremia (CHM) is a rare X-linked disease leading to progressive retinal degeneration resulting in blindness. The disorder is caused by mutations in the CHM gene encoding REP-1 protein, an essential component of the Rab geranylgeranyltransferase (GGTase) complex. In the present study, we evaluated a multi-technique analysis algorithm to describe the mutational spectrum identified in a large cohort of cases and further correlate CHM variants with phenotypic characteristics and biochemical defects of choroideremia patients. Molecular genetic testing led to the characterization of 36 out of 45 unrelated CHM families (80%), allowing the clinical reclassification of four CHM families. Haplotype reconstruction showed independent origins for the recurrent p.Arg293* and p.Lys178Argfs*5 mutations, suggesting the presence of hotspots in CHM, as well as the identification of two different unrelated events involving exon 9 deletion. No certain genotype-phenotype correlation could be established. Furthermore, all the patients´ fibroblasts analyzed presented significantly increased levels of unprenylated Rabs proteins compared to control cells; however, this was not related to the genotype. This research demonstrates the major potential of the algorithm proposed for diagnosis. Our data enhance the importance of establish a differential diagnosis with other retinal dystrophies, supporting the idea of an underestimated prevalence of choroideremia. Moreover, they suggested that the severity of the disorder cannot be exclusively explained by the genotype.

Retinitis pigmentosa (RP) is a group of inherited progressive retinal dystrophies (RD) characterized by photoreceptor degeneration. RP is highly heterogeneous both clinically and genetically, which complicates the identification of causative genes and mutations. Targeted next-generation sequencing (NGS) has been demonstrated to be an effective strategy for the detection of mutations in RP. In our study, an in-house gene panel comprising 75 known RP genes was used to analyze a cohort of 47 unrelated Spanish families pre-classified as autosomal recessive or isolated RP. Disease-causing mutations were found in 27 out of 47 cases achieving a mutation detection rate of 57.4%. In total, 33 pathogenic mutations were identified, 20 of which were novel mutations (60.6%). Furthermore, not only single nucleotide variations but also copy-number variations, including three large deletions in the USH2A and EYS genes, were identified. Finally seven out of 27 families, displaying mutations in the ABCA4, RP1, RP2 and USH2A genes, could be genetically or clinically reclassified. These results demonstrate the potential of our panel-based NGS strategy in RP diagnosis.

Inherited retinal dystrophies present extensive phenotypic and genetic heterogeneity, posing a challenge for patients’ molecular and clinical diagnoses. In this study, we wanted to clinically characterize and investigate the molecular etiology of an atypical form of autosomal recessive retinal dystrophy in two consanguineous Spanish families. Affected members of the respective families exhibited an array of clinical features including reduced visual acuity, photophobia, defective color vision, reduced or absent ERG responses, macular atrophy and pigmentary deposits in the peripheral retina. Genetic investigation included autozygosity mapping coupled with exome sequencing in the first family, whereas autozygome-guided candidate gene screening was performed by means of Sanger DNA sequencing in the second family. Our approach revealed nucleotide changes in CDHR1; a homozygous missense variant (c.1720C > G, p.P574A) and a homozygous single base transition (c.1485 + 2T > C) affecting the canonical 5’ splice site of intron 13, respectively. Both changes co-segregated with the disease and were absent among cohorts of unrelated control individuals. To date, only five mutations in CDHR1 have been identified, all resulting in premature stop codons leading to mRNA nonsense mediated decay. Our work reports two previously unidentified homozygous mutations in CDHR1 further expanding the mutational spectrum of this gene.

Retinitis pigmentosa (RP) is a group of progressive inherited retinal dystrophies that cause visual impairment as a result of photoreceptor cell death. RP is heterogeneous, both clinically and genetically making difficult to establish precise genotype–phenotype correlations. In a Spanish family with autosomal recessive RP (arRP), homozygosity mapping and whole-exome sequencing led to the identification of a homozygous mutation (c.358_359delGT; p.Ala122Leufs*2) in the ZNF408 gene. A screening performed in 217 additional unrelated families revealed another homozygous mutation (c.1621C>T; p.Arg541Cys) in an isolated RP case. ZNF408 encodes a transcription factor that harbors 10 predicted C2H2-type fingers thought to be implicated in DNA binding. To elucidate the ZNF408 role in the retina and the pathogenesis of these mutations we have performed different functional studies. By immunohistochemical analysis in healthy human retina, we identified that ZNF408 is expressed in both cone and rod photoreceptors, in a specific type of amacrine and ganglion cells, and in retinal blood vessels. ZNF408 revealed a cytoplasmic localization and a nuclear distribution in areas corresponding with the euchromatin fraction. Immunolocalization studies showed a partial mislocalization of the p.Arg541Cys mutant protein retaining part of the WT protein in the cytoplasm. Our study demonstrates that ZNF408, previously associated with Familial Exudative Vitreoretinopathy (FEVR), is a new gene causing arRP with vitreous condensations supporting the evidence that this protein plays additional functions into the human retina. This work is supported by CIBERER (06/07/0036), FIS (PI013/00226), Ministry of Economy and Competitiveness-FEDER (BFU2012-36845), RETICS (RD12/0034/0010), Fundación ONCE, Fundaluce and grants BIO2011-27069 from the Spanish Ministry of Economy and Competitiveness, and PROMETEOII/2014/025 from the Conselleria de Educacio of the Valencia Community. PC is supported by Fundación Conchita Rábago (FCR), MC by Miguel Servet ISCIII (CP/03256) and dS by CAPES Foundation, Ministry of Education of Brazil.

PURPOSE The aim of this study was to deepen our knowledge on the basis of intrafamilial genetic heterogeneity of inherited retinal dystrophies (RD) to further discern the contribution of individual alleles to the pathology. METHODS Families with intrafamilial locus and/or allelic heterogeneity were selected from a cohort of 873 characterized of 2468 unrelated RD families. Clinical examination included visual field assessments, electrophysiology, fundus examination, and audiogram. Molecular characterization was performed using a combination of different methods: genotyping microarray, single strand conformational polymorphism (SSCP), denaturing high pressure liquid chromatography (dHPLC), high resolution melt (HRM), multiplex ligation-dependent probe amplification (MLPA), Sanger sequencing, whole-genome homozygosity mapping, and next-generation sequencing (NGS). RESULTS Overall, intrafamilial genetic heterogeneity was encountered in a total of 8 pedigrees. There were 5 of 873 families (~0.6%) with causative mutations in more than one gene (locus heterogeneity), involving the genes: (1) USH2A, RDH12, and TULP1; (2) PDE6B and a new candidate gene; (3) CERKL and CRB1; (4) BBS1 and C2orf71; and (5) ABCA4 and CRB1. Typically, in these cases, each mutated gene was associated with different phenotypes. In the 3 other families (~0.35%), different mutations in the same gene (allelic heterogeneity) were found, including the frequent RD genes ABCA4 and CRB1. CONCLUSIONS This systematic research estimates that the frequency of overall mutation load promoting RD intrafamilial heterogeneity in our cohort of Spanish families is almost 1%. The identification of the genetic mechanisms underlying RD locus and allelic heterogeneity is essential to discriminate the real contribution of the monoallelic mutations to the disease, especially in the NGS era. Moreover, it is decisive to provide an accurate genetic counseling and in disease treatment.

PURPOSE Next-generation sequencing (NGS) has been demonstrated to be an effective strategy for the detection of mutations in retinal dystrophies, a group of inherited diseases that are highly heterogeneous. Therefore, the aim of this study is the application of an NGS-based approach in a Spanish cohort of autosomal dominant retinitis pigmentosa (RP) patients to find out causative mutations. METHODS Index cases of 59 Spanish families with initial diagnosis of autosomal dominant RP and unsuccessfully studied for mutations in the most common RP causal genes, were selected for application of a NGS-based approach with a custom panel for 73 genes related to retinal dystrophies. Candidate variants were select based on frequency, pathogenicity, inherited model, and phenotype. Subsequently, confirmation by Sanger sequencing, cosegregation analysis, and population studies, was applied for determining the implication of those variants in the pathology. RESULTS Overall 31 candidate variants were selected. From them, 17 variants were considered as mutations causative of the disease, 64% (11/17) of them were novel and 36% (6/17) were known RP-related mutations. Therefore, applying this technology16 families were characterized rendering a mutation detection rate of 27% (16/59). Of them, 5% (3/59) of cases displayed mutations in recessive or X-linked genes (ABCA4, RPGR, and RP2) allowing a genetic and clinical reclassification of those families. Furthermore, seven novel variants with uncertain significance and seven novel variants probably not causative of disease were also found. CONCLUSIONS This NGS strategy is a fast, effective, and reliable tool to detect known and novel mutations in autosomal dominant RP patients allowing genetic reclassification in some cases and increasing the knowledge of pathogenesis in retinal dystrophies.

Importance The families evaluated in this study represent the second report of cone-rod dystrophy (CRD) cases caused by mutations inRAB28, a recently discovered gene associated with CRD. Objective To determine the disease-causing gene in 2 families of Spanish descent presenting with CRD who do not haveABCA4mutations. Design, Setting, and Participants Molecular genetics and observational case studies of 2 families, each with 1 affected proband with CRD and 3 or 5 unaffected family members. The affected individual from each family received a complete ophthalmic examination including assessment of refractive errors and best-corrected visual acuity, biomicroscopy, color fundus photography, electroretinography analysis, and visual-evoked potential analysis. After complete sequencing of theABCA4gene with negative results, the screening for disease-causing mutations was performed by whole-exome sequencing. Possible disease-associated variants were determined by filtering based on minor allele frequency, predicted pathogenicity, and segregation analysis in all family members. Main Outcomes and Measures The appearance of the macula was evaluated by clinical examination, fundus photography, and fundus autofluorescence imaging, and visual function was assessed by electroretinography. Disease-causing mutations were assessed by sequence analyses. Results Ophthalmologic findings included markedly reduced visual acuity, bull’s eye maculopathy, foveal hyperpigmentation, peripapillary atrophy, dyschromatopsia, extinguished photopic responses, and reduced scotopic responses observed on electroretinography consistent with the CRD phenotype often associated withABCA4mutations. Although noABCA4mutations were detected in either patient, whole-exome sequencing analysis identified 2 new homozygous mutations in the recently describedRAB28gene, the c.172 + 1G>C splice site variant in IVS2 and the missense c.T651G:p.C217W substitution. Both variants were determined as deleterious by predictive programs and were segregated with the disease in both families. Sequencing of 107 additional patients of Spanish descent with CRD did not reveal other cases withRAB28mutations. Conclusions and Relevance Deleterious mutations inRAB28result in a classic CRD phenotype and are an infrequent cause of CRD in the Spanish population.