PRPH2-Related Retinal Dystrophies: Mutational Spectrum in 103 Families from a Spanish Cohort.

PRPH2, one of the most frequently inherited retinal dystrophy (IRD)-causing genes, implies a high phenotypic variability. This study aims to analyze the PRPH2 mutational spectrum in one of the largest cohorts worldwide, and to describe novel pathogenic variants and genotype-phenotype correlations. A study of 220 patients from 103 families recruited from a database of 5000 families. A molecular diagnosis was performed using classical molecular approaches and next-generation sequencing. Common haplotypes were ascertained by analyzing single-nucleotide polymorphisms. We identified 56 variants, including 11 novel variants. Most of them were missense variants (64%) and were located in the D2-loop protein domain (77%). The most frequently occurring variants were p.Gly167Ser, p.Gly208Asp and p.Pro221_Cys222del. Haplotype analysis revealed a shared region in families carrying p.Leu41Pro or p.Pro221_Cys222del. Patients with retinitis pigmentosa presented an earlier disease onset. We describe the largest cohort of IRD families associated with PRPH2 from a single center. Most variants were located in the D2-loop domain, highlighting its importance in interacting with other proteins. Our work suggests a likely founder effect for the variants p.Leu41Pro and p.Pro221_Cys222del in our Spanish cohort. Phenotypes with a primary rod alteration presented more severe affectation. Finally, the high phenotypic variability in PRPH2 hinders the possibility of drawing genotype-phenotype correlations.

Comprehensive Genotyping and Phenotyping Analysis of GUCY2D-Associated Rod- and Cone-Dominated Dystrophies.

PURPOSE: To describe the genetic and clinical spectrum of GUCY2D-associated retinopathies and to accurately establish their prevalence in a large cohort of patients. DESIGN: Retrospective case series. METHODS: Institutional study of 47 patients from 27 unrelated families with retinal dystrophies carrying disease-causing GUCY2D variants from the Fundación Jiménez Díaz hospital dataset of 8000 patients. Patients underwent ophthalmological examination and molecular testing by Sanger or exome sequencing approaches. Statistical and principal component analyses were performed to determine genotype-phenotype correlations. RESULTS: Four clinically different associated phenotypes were identified: 66.7% of families with cone/cone-rod dystrophy, 22.2% with Leber congenital amaurosis, 7.4% with early-onset retinitis pigmentosa, and 3.7% with congenital night blindness. Twenty-three disease-causing GUCY2D variants were identified, including 6 novel variants. Biallelic variants accounted for 28% of patients, whereas most carried dominant alleles associated with cone/cone-rod dystrophy. The disease onset had statistically significant differences according to the functional variant effect. Patients carrying GUCY2D variants were projected into 3 subgroups by allelic combination, disease onset, and presence of nystagmus or night blindness. In contrast to patients with the most severe phenotype of Leber congenital amaurosis, 7 patients with biallelic GUCY2D had a later and milder rod form with night blindness in infancy as the first symptom. CONCLUSIONS: This study represents the largest GUCY2D cohort in which 4 distinctly different phenotypes were identified, including rare intermediate presentations of rod-dominated retinopathies. We established that GUCY2D is linked to about 1% of approximately 3000 molecularly characterized families of our cohort. All of these findings are critical for defining cohorts for inclusion in future clinical trials.

Application of multicolour reflectance imaging for the characterisation of inherited retinal disorders.

AIM: To describe the role of multicolour reflectance images (MCI) in the phenotypic diagnosis of inherited retinal disorders (IRDs). METHODS: A retrospective review of consecutive patients affected by IRDs examined with MCI techniques from January to December 2019 at a tertiary care referral centre. All patients had MCI, fundus autofluorescence and optical coherence tomography taken at the same time point. The ability of each modality to highlight clinical features was assessed. Lesions' size was also measured and compared among imaging modalities. RESULTS: Thirty eyes of 15 patients were included in the study, 6 males and 9 females, with a mean age of 44 years (range: 19-57.5). The most frequent clinical diagnosis were: pattern dystrophies, and late-onset retinal degeneration. Next-generation or Sanger sequencing analysis was carried out in all patients. Blue and green reflectance were relevant in highlighting peripheral mottling in fundus albipunctatus, pseudoreticular drusen in late-onset retinal degeneration, parafoveal hyperreflective area in bull's eye maculopathy and crystals in Bietti's crystalline dystrophy. Likewise, it is to mention the ability of infrared reflectance to detect hyperreflective patches in posterior pole in neurofibromatosis type 1 and retinal changes in pattern dystrophies and cone dystrophies. CONCLUSION: Multicolour imaging technique enables the detection of clinical features that could be overlooked by other imaging modalities, allowing accurate phenotypic characterisation of IRDs and guiding genetic diagnose, and may become a meaningful monitoring tool for future treatments.

Aggregated Genomic Data as Cohort-Specific Allelic Frequencies can Boost Variants and Genes Prioritization in Non-Solved Cases of Inherited Retinal Dystrophies.

The introduction of NGS in genetic diagnosis has increased the repertoire of variants and genes involved and the amount of genomic information produced. We built an allelic-frequency (AF) database for a heterogeneous cohort of genetic diseases to explore the aggregated genomic information and boost diagnosis in inherited retinal dystrophies (IRD). We retrospectively selected 5683 index-cases with clinical exome sequencing tests available, 1766 with IRD and the rest with diverse genetic diseases. We calculated a subcohort's IRD-specific AF and compared it with suitable pseudocontrols. For non-solved IRD cases, we prioritized variants with a significant increment of frequencies, with eight variants that may help to explain the phenotype, and 10/11 of uncertain significance that were reclassified as probably pathogenic according to ACMG. Moreover, we developed a method to highlight genes with more frequent pathogenic variants in IRD cases than in pseudocontrols weighted by the increment of benign variants in the same comparison. We identified 18 genes for further studies that provided new insights in five cases. This resource can also help one to calculate the carrier frequency in IRD genes. A cohort-specific AF database assists with variants and genes prioritization and operates as an engine that provides a new hypothesis in non-solved cases, augmenting the diagnosis rate.

Allelic overload and its clinical modifier effect in Bardet-Biedl syndrome.

Bardet-Biedl syndrome (BBS) is an autosomal recessive ciliopathy characterized by extensive inter- and intra-familial variability, in which oligogenic interactions have been also reported. Our main goal is to elucidate the role of mutational load in the clinical variability of BBS. A cohort of 99 patients from 77 different families with biallelic pathogenic variants in a BBS-associated gene was retrospectively recruited. Human Phenotype Ontology terms were used in the annotation of clinical symptoms. The mutational load in 39 BBS-related genes was studied in index cases using different molecular and next-generation sequencing (NGS) approaches. Candidate allele combinations were analysed using the in silico tools ORVAL and DiGePred. After clinical annotation, 76 out of the 99 cases a priori fulfilled established criteria for diagnosis of BBS or BBS-like. BBS1 alleles, found in 42% of families, were the most represented in our cohort. An increased mutational load was excluded in 41% of the index cases (22/54). Oligogenic inheritance was suspected in 52% of the screened families (23/45), being 40 tested by means of NGS data and 5 only by traditional methods. Together, ORVAL and DiGePred platforms predicted an oligogenic effect in 44% of the triallelic families (10/23). Intrafamilial variable severity could be clinically confirmed in six of the families. Our findings show that the presence of more than two alleles in BBS-associated genes correlated in six families with a more severe phenotype and associated with specific findings, highlighting the role of the mutational load in the management of BBS cases.

Impact of Next Generation Sequencing in Unraveling the Genetics of 1036 Spanish Families With Inherited Macular Dystrophies.

Purpose: To assess the potential of next-generation sequencing (NGS) technologies to characterize cases diagnosed with autosomal recessive (ar) or sporadic (s) macular dystrophies (ar/sMD) and describe their mutational spectrum. Methods: A cohort of 1036 families was classified according to their suspected clinical diagnosis-Stargardt disease (STGD), cone and cone-rod dystrophy (CCRD) or other maculopathies (otherMD). Molecular studies included genotyping microarrays, Sanger sequencing, NGS, and sequencing of intronic regions of the ABCA4 gene. Clinical reclassification was done after the genetic study. Results: At the end of the study, 677 patients (65%) had a confirmed genetic diagnosis, representing 78%, 63%, and 38% of STGD, CCRD, and otherMD groups of patients, respectively. ABCA4 is the most mutated gene in all groups, and a second pathogenic variant was found in 76% of STGD patients with one previously identified mutated ABCA4 allele. Autosomal dominant or X-linked mutations were found in 5% of cases together with not-MD genes (CHM, EYS, RHO, RPGR, RLBP1, OPA1, and USH2A among others) leading to their reclassification. Novel variants in the very rare genes PLA2G5 and TTLL5 revealed additional phenotypic associations. Conclusions: This study provides for the first time a genetic landscape of 1036 ar/sMD families according to their suspected diagnosis. The analysis of >200 genes associated with retinal dystrophies and the entire locus of ABCA4 increase the rate of characterization, even regardless of available clinical and familiar data. The use of the suspected a priori diagnosis referred by the clinicians, especially in the past, could lead to clinical reclassifications to other inherited retinal dystrophies.

An evaluation of pipelines for DNA variant detection can guide a reanalysis protocol to increase the diagnostic ratio of genetic diseases.

Clinical exome (CE) sequencing has become a first-tier diagnostic test for hereditary diseases; however, its diagnostic rate is around 30-50%. In this study, we aimed to increase the diagnostic yield of CE using a custom reanalysis algorithm. Sequencing data were available for three cohorts using two commercial protocols applied as part of the diagnostic process. Using these cohorts, we compared the performance of general and clinically relevant variant calling and the efficacy of an in-house bioinformatic protocol (FJD-pipeline) in detecting causal variants as compared to commercial protocols. On the whole, the FJD-pipeline detected 99.74% of the causal variants identified by the commercial protocol in previously solved cases. In the unsolved cases, FJD-pipeline detects more INDELs and non-exonic variants, and is able to increase the diagnostic yield in 2.5% and 3.2% in the re-analysis of 78 cancer and 62 cardiovascular cases. These results were considered to design a reanalysis, filtering and prioritization algorithm that was tested by reassessing 68 inconclusive cases of monoallelic autosomal recessive retinal dystrophies increasing the diagnosis by 4.4%. In conclusion, a guided NGS reanalysis of unsolved cases increases the diagnostic yield in genetic disorders, making it a useful diagnostic tool in medical genetics.

RPE65-related retinal dystrophy: Mutational and phenotypic spectrum in 45 affected patients.

INTRODUCTION: Biallelic pathogenic RPE65 variants are related to a spectrum of clinically overlapping inherited retinal dystrophies (IRD). Most affected individuals progress to severe disease, with 50% of patients becoming legally blind by 20 years of age. Deeper knowledge of the mutational spectrum and the phenotype-genotype correlation in RPE65-related IRD is needed. PATIENTS AND METHODS: Forty-five affected subjects from 27 unrelated families with a clinical diagnosis of RPE65-related IRD were included. Clinical evaluation consisted of self-reported ophthalmological history and objective ophthalmological examination. Patients' genotype was classified according to variant class (truncating or missense) or to variant location at different protein domains. The main phenotypic outcome measure was age at onset (AAO) of symptomatic disease and a Kaplan-Meier analysis of disease symptom event-free survival was performed. RESULTS: Twenty-nine different RPE65 variants were identified in our cohort, 7 of them novel. Patients carrying two missense alleles showed a later disease onset than those with 1 or 2 truncating variants (log-rank test p <0.05). While 60% of patients carrying a missense/missense genotype presented symptoms before or during the first year of life, almost all patients with at least 1 truncating allele (91%) had an AAO ≤1 year (p <0.05). CONCLUSION: Our findings suggest an association between the type of RPE65 variant carried and AAO. These findings provide useful data on RPE65-associated IRD phenotypes and may help improve clinical and therapeutic management of these patients.

Homozygous females for a X-linked RPGR-ORF15 mutation in an Iranian family with retinitis pigmentosa.

Mutations in Retinitis pigmentosa GTPase regulator gene (RPGR) are the most common cause of X-linked retinitis pigmentosa (RP). Almost 60% of disease-causing RPGR mutations are located in ORF-15 region which cannot be detected by Next Generation Sequencing (NGS) due to the existence of highly repetitive regions. An Iranian family with a priori diagnosis of autosomal dominant RP was studied by Sanger sequencing of ORF15 of RPGR gene after an inconclusive NGS result. A frameshift two-base-pair deletion (c.2323_2324del, p.Arg775Glufs*59) in this region was segregating in both affected hemizygous males and affected homozygous females. To our knowledge, this is the first example of homozygous females for RPGR-ORF15 mutations.

Apparent but unconfirmed digenism in an Iranian consanguineous family with syndromic Retinal Disease.

BACKGROUND: Bardet-Biedl syndrome is an autosomal recessive disease characterized by rod-cone dystrophy, postaxial polydactyly, kidney defects, obesity, mental retardation and hypogonadism. Here, we report different genotypes in two Bardet-Biedl syndrome affected sisters with a different clinical phenotype regarding severity. MATERIALS AND METHODS: The proband of the family was examined by Next Generation Sequencing (NGS) using clinical exome and filtering by syndromic and non-syndromic genes associated with retinal dystrophies. RESULTS: Targeted NGS revealed two novel variants in the MKKS and CEP290 genes in homozygosis state in the proband. Segregation analysis revealed the presence of the same MKKS homozygous variant in her younger affected sister but not the CEP290 variant. Both sisters presented different clinical manifestation, at different ages, with a more severe renal and retinal defect in the case of the sister carrying mutations in both genes. Another unaffected sister showed only homozygosity for the CEP290 variant, thus supporting the non-pathogenic role of this mutation in BBS phenotype. CONCLUSIONS: In this study, NGS proved to be a powerful and efficient sequencing method to identify causal variants in different genes. However, it remarks the importance of the segregation analysis and clinical information to establish the pathogenicity of new variants. The two affected sisters present different genotypes and clinical manifestation, suggesting that the novel CEP290 variant could be acting as a modifier, making the phenotype more severe in the sister homozygote for MKKS and CEP290 genes. On the other hand, the difference in the age of both sisters highlight the important role of monitoring disease progression also to confirm the modifier role of genetic variants.

Prioritizing variants of uncertain significance for reclassification using a rule-based algorithm in inherited retinal dystrophies.

Inherited retinal dystrophies (IRD) are a highly heterogeneous group of rare diseases with a molecular diagnostic rate of >50%. Reclassification of variants of uncertain significance (VUS) poses a challenge for IRD diagnosis. We collected 668 IRD cases analyzed by our geneticists using two different clinical exome-sequencing tests. We identified 114 unsolved cases pending reclassification of 125 VUS and studied their genomic, functional, and laboratory-specific features, comparing them to pathogenic and likely pathogenic variants from the same cohort (N = 390). While the clinical exome used did not show differences in diagnostic rate, the more IRD-experienced geneticist reported more VUS (p = 4.07e-04). Significantly fewer VUS were reported in recessive cases (p = 2.14e-04) compared to other inheritance patterns, and of all the genes analyzed, ABCA4 and IMPG2 had the lowest and highest VUS frequencies, respectively (p = 3.89e-04, p = 6.93e-03). Moreover, few frameshift and stop-gain variants were found to be informed VUS (p = 6.73e-08 and p = 2.93e-06). Last, we applied five pathogenicity predictors and found there is a significant proof of deleteriousness when all score for pathogenicity in missense variants. Altogether, these results provided input for a set of rules that correctly reclassified ~70% of VUS as pathogenic in validation datasets. Disease- and setting-specific features influence VUS reporting. Comparison with pathogenic and likely pathogenic variants can prioritize VUS more likely to be reclassified as causal.

Genetic landscape of 6089 inherited retinal dystrophies affected cases in Spain and their therapeutic and extended epidemiological implications.

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 performed to the 3951 families with available DNA using different molecular techniques. Overall, 53.2% (2100/3951) of the studied families were genetically characterized, and 1549 different likely causative variants in 142 genes were identified. The most common phenotype encountered is retinitis pigmentosa (RP) (55.6% of families, 2447/4403). The most recurrently mutated genes 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.

Genotype-Phenotype Correlations in a Spanish Cohort of 506 Families With Biallelic ABCA4 Pathogenic Variants.

PURPOSE: To define genotype-phenotype correlations in the largest cohort study worldwide of patients with biallelic ABCA4 variants, including 434 patients with Stargardt disease (STGD1) and 72 with cone-rod dystrophy (CRD). DESIGN: Cohort study. METHODS: We characterized 506 patients with ABCA4 variants using conventional genetic tools and next-generation sequencing technologies. Medical history and ophthalmologic data were obtained from 372 patients. Genotype-phenotype correlation studies were carried out for the following variables: variant type, age at symptom onset (AO), and clinical phenotype. RESULTS: A total of 228 different pathogenic variants were identified in 506 ABCA4 patients, 50 of which were novel. Genotype-phenotype correlations showed that most of the patients with biallelic truncating variants presented with CRD and that these cases had a significantly earlier AO than patients with STGD1. Three missense variants are associated with CRD for the first time (c.1804C>T; p.[Arg602Trp], c.3056C>T; p.[Thr1019Met], and c.6320G>C; p.[Arg2107Pro]). Analysis of the most prevalent ABCA4 variant in Spain, c.3386G>T; p.(Arg1129Leu), revealed that is correlated to STGD1, later AO, and foveal sparing. CONCLUSIONS: Our study, conducted in the largest ABCA4-associated disease cohort reported to date, updates the genotype-phenotype model established for ABCA4 variants and broadens the mutational spectrum of the gene. According to our observations, patients with ABCA4 presenting with 2 truncating variants may first present features of STGD1 but eventually develop rod dysfunction, and specific missense variants may be associated with a different phenotype, underscoring the importance of an accurate genetic diagnosis. Also, it is a prerequisite for enrollment in clinical trials, and to date, no other treatment has been approved for STGD1.

Seudohiperfosfatemia analítica como punto de partida en el diagnóstico de una disproteinemia / Role of pseudo-hyperphosphataemia in the diagnosis of dysproteinaemia

El fósforo es el segundo mineral más abundante en el organismo. Su homeostasis se consigue mantener a través de varios mecanismos mediados principalmente por el riñón, el intestino y el hueso. Se han descrito interferencias en la medición del fósforo que pueden provocar una seudohiperfosfatemia. La causa más frecuente es la presencia de una paraproteína en el suero de los pacientes con mieloma múltiple, macroglobulinemia de Waldenström y gammapatía monoclonal de significado incierto. En los casos de hiperfosfatemia sin causa aparente que la pueda justificar, es importante tener en cuenta la existencia de una seudohiperfosfatemia causada por la presencia de las paraproteínas en sangre en los autoanalizadores de química líquida. El sistema multicapa de Vitros(R) 5600 es un método rápido y fiable para solucionar este problema

Phosphorus is the second most important mineral in the body. Its homeostasis is maintained through several mechanisms mediated mainly by the kidney, intestine, and bone. Interferences have been described in the measurement of phosphorus that could suggest a pseudo-hyperphosphataemia. The most frequent cause was the presence of a paraprotein in the serum of patients with multiple myeloma, Waldenström macroglobulinaemia, or monoclonal gammopathy of uncertain significance, was described as the most frequent cause of interference in phosphorus assay using liquid chemistry autoanalysers. When hyperphosphataemia is present, and no apparent cause can justify it, it is important to consider the possibility of a pseudo-hyperphosphataemia caused mainly by the presence of a paraprotein. The Vitros(R) 5600 multilayer system can be used as a fast and reliable method to avoid this interference

Expanded Phenotypic Spectrum of Retinopathies Associated with Autosomal Recessive and Dominant Mutations in PROM1.

PURPOSE: To describe the genetic and phenotypic characteristics of a cohort of patients with PROM1 variants. DESIGN: Case-case study. METHODS: We screened a cohort of 2216 families with inherited retinal dystrophies using classical molecular techniques and next-generation sequencing approaches. The clinical histories of 25 patients were reviewed to determine age of onset of symptoms and the results of ophthalmoscopy, best-corrected visual acuity, full-field electroretinography, and visual field studies. Fundus autofluorescence and spectral-domain optical coherence tomography were further assessed in 7 patients. RESULTS: PROM1 variants were identified in 32 families. Disease-causing variants were found in 18 autosomal recessive and 4 autosomal dominant families. Monoallelic pathogenic variants or variants of unknown significance were identified in the remaining 10 families. Comprehensive phenotyping of 25 patients from 22 families carrying likely disease-causing variants revealed clinical heterogeneity associated with the PROM1 gene. Most of these patients presented cone-rod dystrophy and some exhibited macular dystrophy or retinitis pigmentosa, while all presented with macular damage. Phenotypic association of a dominant splicing variant with late-onset mild maculopathy was established. This variant is one of the 3 likely founder variants identified in our Spanish cohort. CONCLUSIONS: We report the largest cohort of patients with PROM1 variants, describing in detail the phenotype in 25 of them. Interestingly, within the variability of phenotypes related to this gene, macular involvement is a common feature in all patients.

Genomic Landscape of Sporadic Retinitis Pigmentosa: Findings from 877 Spanish Cases.

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.

Unravelling the pathogenic role and genotype-phenotype correlation of the USH2A p.(Cys759Phe) variant among Spanish families.

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≤0.05) in age at diagnosis of RP and hypoacusis, and progression of visual field loss. Homozygosity of p.(Cys759Phe) and compound heterozygosity with another USH2A missense variant is associated with ARRP or ARRP plus late onset hypoacusis (OR = 20.62, CI = 95%, p = 0.041). CONCLUSIONS: The present study supports the role of USH2A p.(Cys759Phe) in ARRP and USH2 pathogenesis, and demonstrates the clinical differences between different zygosity states. Phenotype-genotype correlations may guide the genetic characterization based upon specific clinical signs and may advise on the clinical management and prognosis based upon a specific genotype.

Toward the Mutational Landscape of Autosomal Dominant Retinitis Pigmentosa: A Comprehensive Analysis of 258 Spanish Families.

Purpose: To provide a comprehensive overview of the molecular basis of autosomal dominant retinitis pigmentosa (adRP) in Spanish families. Thus, we established the molecular characterization rate, gene prevalence, and mutational spectrum in the largest European cohort reported to date. Methods: A total of 258 unrelated Spanish families with a clinical diagnosis of RP and suspected autosomal dominant inheritance were included. Clinical diagnosis was based on complete ophthalmologic examination and family history. Retrospective and prospective analysis of Spanish adRP families was carried out using a combined strategy consisting of classic genetic techniques and next-generation sequencing (NGS) for single-nucleotide variants and copy number variation (CNV) screening. Results: Overall, 60% of our families were genetically solved. Interestingly, 3.1% of the cohort carried pathogenic CNVs. Disease-causing variants were found in an autosomal dominant gene in 55% of the families; however, X-linked and autosomal recessive forms were also identified in 3% and 2%, respectively. Four genes (RHO, PRPF31, RP1, and PRPH2) explained up to 62% of the solved families. Missense changes were most frequently found in adRP-associated genes; however, CNVs represented a relevant disease cause in PRPF31- and CRX-associated forms. Conclusions: Implementation of NGS technologies in the adRP study clearly increased the diagnostic yield compared with classic approaches. Our study outcome expands the spectrum of disease-causing variants, provides accurate data on mutation gene prevalence, and highlights the implication of CNVs as important contributors to adRP etiology.