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To enhance the use of Whole Genome Sequencing (WGS) in clinical practice, it is still necessary to standardize data analysis pipelines. Herein, we aimed to define a WGS-based algorithm for the accurate interpretation of variants in inherited retinal dystrophies (IRD). This study comprised 429 phenotyped individuals divided into three cohorts. A comparison of 14 pathogenicity predictors, and the re-definition of its cutoffs, were performed using panel-sequencing curated data from 209 genetically diagnosed individuals with IRD (training cohort). The optimal tool combinations, previously validated in 50 additional IRD individuals, were also tested in patients with hereditary cancer (n = 109), and with neurological diseases (n = 47) to evaluate the translational value of this approach (validation cohort). Then, our workflow was applied for the WGS-data analysis of 14 individuals from genetically undiagnosed IRD families (discovery cohort). The statistical analysis showed that the optimal filtering combination included CADDv1.6, MAPP, Grantham, and SIFT tools. Our pipeline allowed the identification of one homozygous variant in the candidate gene CFAP20 (c.337 C > T; p.Arg113Trp), a conserved ciliary gene, which was abundantly expressed in human retina and was located in the photoreceptors layer. Although further studies are needed, we propose CFAP20 as a candidate gene for autosomal recessive retinitis pigmentosa. Moreover, we offer a translational strategy for accurate WGS-data prioritization, which is essential for the advancement of personalized medicine.

To enhance the use of Whole Genome Sequencing (WGS) in clinical practice, it is still necessary to standardize data analysis pipelines. Herein, we aimed to define a WGS-based algorithm for the accurate interpretation of variants in inherited retinal dystrophies (IRD). This study comprised 429 phenotyped individuals divided into three cohorts. A comparison of 14 pathogenicity predictors, and the re-definition of its cutoffs, were performed using panel-sequencing curated data from 209 genetically diagnosed individuals with IRD (training cohort). The optimal tool combinations, previously validated in 50 additional IRD individuals, were also tested in patients with hereditary cancer (n = 109), and with neurological diseases (n = 47) to evaluate the translational value of this approach (validation cohort). Then, our workflow was applied for the WGS-data analysis of 14 individuals from genetically undiagnosed IRD families (discovery cohort). The statistical analysis showed that the optimal filtering combination included CADDv1.6, MAPP, Grantham, and SIFT tools. Our pipeline allowed the identification of one homozygous variant in the candidate gene CFAP20 (c.337 C > T; p.Arg113Trp), a conserved ciliary gene, which was abundantly expressed in human retina and was located in the photoreceptors layer. Although further studies are needed, we propose CFAP20 as a candidate gene for autosomal recessive retinitis pigmentosa. Moreover, we offer a translational strategy for accurate WGS-data prioritization, which is essential for the advancement of personalized medicine.

[Purpose] To describe the clinical and genetic characteristics (novel mutation in BEST1 gene) of a Spanish patient with autosomal recessive bestrophinopathy (ARB)., [Methods] The detailed ophthalmological examination included best corrected visual acuity (BCVA), color and autofluorescence photography, fluorescein angiography, optical coherence tomography, and electrophysiology tests. A next-generation sequencing (NGS) strategy was applied to the index patient, and then sequenced in an Illumina NextSeq500 system., [Results] A 55-year-old male presented with a BCVA of 20/25 in the right eye and 20/20 in the left eye. Fundoscopy revealed perifoveal yellow flecked-like lesions. Fluorescein angiography and fundus autofluorescence results were consistent with pattern dystrophy. A homozygous frameshift mutation in BEST1 (c.341_342del; p.(Leu114Glnfs*57)) was identified as the cause of the disease., [Conclusion] ARB is a genetic disease that leads to irreversible visual loss. In this report we found a novel mutation responsible for this disease.

Purpose: Artificial tears only provide transient relief for dry eye. To the best of our knowledge, this is the first study to objectively compare treatment with artificial tears with Keratograph 5M, which allows accurate and objective investigation of dry eye and artificial tear treatment. We aimed to evaluate whether a preservative-free combination of 0.4% hyaluronic acid and 0.2% galactoxyloglucan can improve dry eye using the new topographer, Keratograph 5M. Patients and Methods: This prospective longitudinal, single-arm interventional cohort study was performed at a tertiary referral center and involved 20 patients with dry eye (40 eyes). Preservative-free artificial tears were administered every 3 h. The participants under went clinical and instrumental evaluations at baseline, 15, 30, 60, 90 and 120 min after instillation and 1 week and 1 month after treatment. Baseline values were considered as the controls. All patients were assessed with Keratograph 5M for non-invasive Keratograph first break-up time and non-invasive Keratograph average break-up time, conjunctival hyperemia, and tear meniscus height. Ocular surface staining with fluorescein was evaluated using the slit-lamp and fluorescein break-up time examinations, and the Ocular Surface Disease Index score was recorded for each patient. Results: The signs and symptoms improved after 1 month of preservative-free 0.4% hyaluronic acid and 0.2% galactoxyloglucan treatment. There was a significant increase in the non-invasive Keratograph first break-up time and non-invasive Keratograph average break-up time at 15, 30, 60, and 90 min, and 1 week and 1 month (P < 0.05) and a decrease in hyperemia, corneal staining, and Ocular Surface Disease Index scores after 1 week and 1 month (P < 0.05). No treatment-related adverse event was observed. Conclusion: A combination of 0.4% hyaluronic acid and 0.2% galactoxyloglucan artificial tears seems effective for treating dry eye. Keratograph 5M can objectively detect these

Background: We report a Spanish family, comprising an affected mother and daughter, respectively diagnosed with retinitis pigmentosa and Stargardt-like macular dystrophy, in whom we identified a PROM1 mutation. Methods: A custom gene panel consisting of 119 inherited retinal dystrophies (IRD)-genes was applied in the two affected individuals of this family and sequenced using the Illumina ́s NextSeq 500 platform. Results: The analysis of the resulting data allowed us to identify the pathogenic PROM1 mutation c.1117C>T (p.Arg373Cys) as the primary cause of the disease in both patients. No additional variants contributing to the extent of retinal dysfunction were detected. Conclusion: The variable expressivity of the detected PROM1 mutation is the most likely responsible for the intrafamilial phenotypic variability observed inthis family. Screening of this mutation should be considered in patients with compatible clinical manifestations, especially when accompanied by an autosomal dominant family history.

Mutations in the ABCA4 gene are a common cause of Stargardt disease; however, other retinal phenotypes have also been associated with mutations in this gene. We describe an observational case report of an unusual clinical phenotype of Stargardt disease. The ophthalmological examination included best corrected visual acuity, color and autofluorescence photography, fluorescein angiography, optical coherence tomography, and electrophysiology tests. Targeted next-generation sequencing of 99 genes associated with inherited retinal dystrophies was performed in the index patient. A 48-year-old woman presented with a best corrected visual acuity of 20/25 and 20/20. Fundoscopy revealed perifoveal yellow flecked-like lesions. Fluorescein angiography and fundus autofluorescence findings were consistent with pattern dystrophy. Pattern electroretinogram demonstrated bilateral decrease of p50 values. Genetic testing identified two heterozygous missense mutations, c.428C>T, p.(Pro143Leu) and c.3113C>T, p.(Ala.1038Val), in the ABCA4 gene. Based on our results, we believe that these particular mutations in the ABCA4 gene could be associated with a specific disease phenotype characterized by funduscopic appearance similar to pattern dystrophy. A detailed characterization of the retinal phenotype in patients carrying specific mutations in ABCA4 is crucial to understand disease expression and ensure optimal clinical care for patients with inherited retinal dystrophies., Mutações no gene ABCA4 são causa comum da doença de Stargardt, mas outros fenótipos da retina também foram associados a mutações nesse gene. Apresentamos um relato de caso observacional de um fenótipo clínico incomum da doença de Stargardt. O exame oftalmológico incluiu a acuidade visual com melhor correção, fotografia em cores e com autofluores cência, angiofluoresceinografia, tomografia de coerência óptica e testes de eletrofisiologia. Na paciente em questão, realizou-se o sequenciamento de próxima geração de 99 genes associados a distrofias retinais hereditárias. Tratava-se de uma mulher de 48 anos com melhor acuidade visual corrigida de 20/25 e 20/20. A fundoscopia revelou lesões puntiformes amarelas perifoveais. Os resultados da angiofluoresceinografia e da autofluorescência do fundo de olho foram consistentes com distrofia em padrão. A eletrorretinografia por padrões mostrou diminuição bilateral dos valores de p50. Os testes genéticos revelaram duas mutações missense heterozigóticas, c.428C>T, p. (Pro143Leu) e c.3113C>T, p. (Ala.1038Val), no gene ABCA4. Nossos resultados nos fazem pensar que essas mutações específicas em ABCA4 talvez possam estar associadas a um fenótipo específico da doença, caracterizado por uma aparência fundoscópica semelhante à da distrofia em padrão. Uma caracterização detalhada do fenótipo da retina em pacientes portadores de mutações específicas em ABCA4 é crucial para compreender a expressão da doença e para garantir o tratamento clínico ideal para pacientes com distrofias retinais hereditárias.

La COVID-19 (enfermedad por coronavirus 2019) nos plantea nuevos retos en nuestra rutina diaria. La última cifra publicada de sanitarios contagiados –se dejó de publicar por última vez el 18 de mayo– fue de 51,090 en total y 152 casos nuevos ese mismo día1. Por ello, debemos aprender a protegernos para continuar con nuestra rutina diaria quirúrgica lo más cercana a la normalidad preexistente.

[EN] Mutations in the ABCA4 gene are a common cause of Stargardt disease; however, other retinal phenotypes have also been associated with mutations in this gene. We describe an observational case report of an unusual clinical phenotype of Stargardt disease. The ophthalmological examination included best corrected visual acuity, color and autofluorescence photography, fluorescein angiography, optical coherence tomography, and electrophysiology tests. Targeted next-generation sequencing of 99 genes associated with inherited retinal dystrophies was performed in the index patient. A 48-year-old woman presented with a best corrected visual acuity of 20/25 and 20/20. Fundoscopy revealed perifoveal yellow flecked-like lesions. Fluorescein angiography and fundus autofluorescence findings were consistent with pattern dystrophy. Pattern electroretinogram demonstrated bilateral decrease of p50 values. Genetic testing identified two heterozygous missense mutations, c.428C>T, p.(Pro143Leu) and c.3113C>T, p.(Ala.1038Val), in the ABCA4 gene. Based on our results, we believe that these particular mutations in the ABCA4 gene could be associated with a specific disease phenotype characterized by funduscopic appearance similar to pattern dystrophy. A detailed characterization of the retinal phenotype in patients carrying specific mutations in ABCA4 is crucial to understand disease expression and ensure optimal clinical care for patients with inherited retinal dystrophies., [PT] Mutações no gene ABCA4 são causa comum da doença de Stargardt, mas outros fenótipos da retina também foram associados a mutações nesse gene. Apresentamos um relato de caso observacional de um fenótipo clínico incomum da doença de Stargardt. O exame oftalmológico incluiu a acuidade visual com melhor correção, fotografia em cores e com autofluorescência, angiofluoresceinografia, tomografia de coerência óptica e testes de eletrofisiologia. Na paciente em questão, realizou-se o sequenciamento de próxima geração de 99 genes associados a distrofias retinais hereditárias. Tratava-se de uma mulher de 48 anos com melhor acuidade visual corrigida de 20/25 e 20/20. A fundoscopia revelou lesões puntiformes amarelas perifoveais. Os resultados da angiofluoresceinografia e da autofluorescência do fundo de olho foram consistentes com distrofia em padrão. A eletrorretinografia por padrões mostrou diminuição bilateral dos valores de p50. Os testes genéticos revelaram duas mutações missense heterozigóticas, c.428C>T, p. (Pro143Leu) e c.3113C>T, p. (Ala.1038Val), no gene ABCA4. Nossos resultados nos fazem pensar que essas mutações específicas em ABCA4 talvez possam estar associadas a um fenótipo específico da doença, caracterizado por uma aparência fundoscópica semelhante à da distrofia em padrão. Uma caracterização detalhada do fenótipo da retina em pacientes portadores de mutações específicas em ABCA4 é crucial para compreender a expressão da doença e para garantir o tratamento clínico ideal para pacientes com distrofias retinais hereditárias.

The management of unsolved inherited retinal dystrophies (IRD) cases is challenging since no standard pipelines have been established. This study aimed to define a diagnostic algorithm useful for the diagnostic routine and to address unsolved cases. Here, we applied a Next-Generation Sequencing-based workflow, including a first step of panel sequencing (PS) followed by clinical-exome sequencing (CES) and whole-exome sequencing (WES), in 46 IRD patients belonging to 42 families. Twenty-six likely causal variants in retinal genes were found by PS and CES. CES and WES allowed proposing two novel candidate loci (WDFY3 and a X-linked region including CITED1), both abundantly expressed in human retina according to RT-PCR and immunohistochemistry. After comparison studies, PS showed the best quality and cost values, CES and WES involved similar analytical efforts and WES presented the highest diagnostic yield. These results reinforce the relevance of panels as a first step in the diagnostic routine and suggest WES as the next strategy for unsolved cases, reserving CES for the simultaneous study of multiple conditions. Standardizing this algorithm would enhance the efficiency and equity of clinical genetics practice. Furthermore, the identified candidate genes could contribute to increase the diagnostic yield and expand the mutational spectrum in these disorders.

[Purpose]: Artificial tears only provide transient relief for dry eye. To the best of our knowledge, this is the first study to objectively compare treatment with artificial tears with Keratograph 5M, which allows accurate and objective investigation of dry eye and artificial tear treatment. We aimed to evaluate whether a preservative-free combination of 0.4% hyaluronic acid and 0.2% galactoxyloglucan can improve dry eye using the new topographer, Keratograph 5M. [Patients and Methods]: This prospective longitudinal, single-arm interventional cohort study was performed at a tertiary referral center and involved 20 patients with dry eye (40 eyes). Preservative-free artificial tears were administered every 3 h. The participants underwent clinical and instrumental evaluations at baseline, 15, 30, 60, 90 and 120 min after instillation and 1 week and 1 month after treatment. Baseline values were considered as the controls. All patients were assessed with Keratograph 5M for non-invasive Keratograph first break-up time and non-invasive Keratograph average break-up time, conjunctival hyperemia, and tear meniscus height. Ocular surface staining with fluorescein was evaluated using the slit-lamp and fluorescein break-up time examinations, and the Ocular Surface Disease Index score was recorded for each patient. [Results]: The signs and symptoms improved after 1 month of preservative-free 0.4% hyaluronic acid and 0.2% galactoxyloglucan treatment. There was a significant increase in the non-invasive Keratograph first break-up time and non-invasive Keratograph average break-up time at 15, 30, 60, and 90 min, and 1 week and 1 month (P < 0.05) and a decrease in hyperemia, corneal staining, and Ocular Surface Disease Index scores after 1 week and 1 month (P < 0.05). No treatment-related adverse event was observed. [Conclusion]: A combination of 0.4% hyaluronic acid and 0.2% galactoxyloglucan artificial tears seems effective for treating dry eye. Keratograph 5M can objectively detec

[Background] Retinitis Pigmentosa (RP) is a clinically and genetically heterogeneous disorder that results in inherited blindness. Despite the large number of genes identified, only ~ 60% of cases receive a genetic diagnosis using targeted-sequencing. The aim of this study was to design a whole genome sequencing (WGS) based approach to increase the diagnostic yield of complex Retinitis Pigmentosa cases., [Methods] WGS was conducted in three family members, belonging to one large apparent autosomal dominant RP family that remained unsolved by previous studies, using Illumina TruSeq library preparation kit and Illumina HiSeq X platform. Variant annotation, filtering and prioritization were performed using a number of open-access tools and public databases. Sanger sequencing of candidate variants was conducted in the extended family members., [Results] We have developed and optimized an algorithm, based on the combination of different open-access tools, for variant prioritization of WGS data which allowed us to reduce significantly the number of likely causative variants pending to be manually assessed and segregated. Following this algorithm, four heterozygous variants in one autosomal recessive gene (USH2A) were identified, segregating in pairs in the affected members. Additionally, two pathogenic alleles in ADGRV1 and PDZD7 could be contributing to the phenotype in one patient., [Conclusions] The optimization of a diagnostic algorithm for WGS data analysis, accompanied by a hypothesis-free approach, have allowed us to unmask the genetic cause of the disease in one large RP family, as well as to reassign its inheritance pattern which implies differences in the clinical management of these cases. These results contribute to increasing the number of cases with apparently dominant inheritance that carry causal mutations in recessive genes, as well as the possible involvement of various genes in the pathogenesis of RP in one patient. Moreover, our WGS-analysis approach, based on open-access tools, can easily be implemented by other researchers and clinicians to improve the diagnostic yield of additional patients with inherited retinal dystrophies.

Purpose: In limbal stem cell deficiency, both the Ocular Surface Disease Index (OSDI) questionnaire and tear break-up time (BUT) are comparable between traditional methods and the Keratograph 5M. In this study, we aimed to correlate OSDI with Keratograph 5M interviewed OSDI, as well as slit-lamp tear BUT with Keratograph 5M noninvasive tear break-up time (NIKBUT) in limbal stem cell deficiency. Patients and methods: Thirty-eight limbal stem cell-deficiency patients (76 eyes) from Virgen Macarena-Rocio Hospital (Seville, Spain) underwent this diagnostic test study. All patients completed the traditional OSDI. We measured the BUT, performed a Keratograph 5M analysis of NIKBUT first (employed for the analysis) followed by the average NIKBUT, the level of dryness, and conducted the OSDI questionnaire through an interview. For each pair of tests, we analyzed the means and applied an intraclass correlation coefficient (r), creating a Bland-Altman plot for data dispersion. Results: Average values were 47.5 points (±25.8), and 47.3 points (±27.5) for traditional OSDI and Keratograph OSDI, respectively (P =0.87); the r value indicates good agreement (0.72). The Bland-Altman plot followed a linear pattern, and the results were similarly distributed. The NIKBUT mean was shorter than the BUT mean (P = 0.007); the r value indicates moderate agreement (0.574). The Bland-Altman plot formed an almost horizontal line, with almost all values between the mean and two standard deviations. Conclusion: Keratograph 5M is useful for the evaluation of the ocular surface in limbal stem cell deficiency. NIKBUT can substitute BUT based on its advantages of being non- invasive, objective, with intraobserver and interobserver repeatability and reliability. The Keratograph 5M OSDI is comparable to the traditional questionnaire.

[Aim] To assess the relationship between choroidal thickness and renal function in diabetic patients., [Methods] Cross-sectional retrospective clinical study of 42 eyes of 21 ocular treatment-na?ve diabetic patients. Demographic data included: age, sex, type and course of diabetes. Ocular data included: severity of diabetic retinopathy; retinal thickness at the central macular region, as well as choroidal thickness at the central and paracentral quadrants, using automatically generated maps by swept-source optical coherence tomography; presence of cystic macular edema; and ocular axial length (AXL). Lab-test parameters included: glycated hemoglobin (HbA1c), albuminuria, albumin/creatinine ratio in urine, and glomerular filtration rate., [Results] A significant negative correlation was mainly observed between several choroidal thicknesses, age (P<0.020) and ocular AXL (P<0.030). On the contrary, a significant positive correlation was found between all choroidal thicknesses, HbA1c (P<0.035) and albuminuria (P<0.040)., [Conclusion] Choroidal thickness can represent an additional tool to help clinicians predicting the renal status in ocular treatment-naïve diabetic patients.

[Background/aims] Limbal stem cell deficiency (LSCD) is characterised by a marked decrease in limbal stem cells. It is classified primarily using subjective slit-lamp observations. In vivo confocal microscopy (IVCM) can non-invasively provide objective information on the condition of the limbal niche, the corneal epithelial basal cell density and the corneal sub-basal nerve plexus density (SND). We here used IVCM to evaluate changes in SND to improve LSCD classification. [Methods] We evaluated and classified 38 patients (76 eyes, 44 with LSC and 32 control eyes) using the Rama, López-García and Deng (clinical and confocal) classifications and evaluated the concordance of the confocal and clinical classifications. We constructed a logistic regression model using multivariate analysis to correlate different degrees of conjunctivalisation with IVCM parameters and used receiver operating characteristic (ROC) curve analysis to establish the SND cut-off value with maximum diagnostic sensitivity and specificity. [Results] The classification systems correlated moderately at best (kappa, 0.449). The corneal SND of cases (6469±6295 µm/mm2) was less (p<0.001) than in controls (20911±4142 µm/mm2). The SND, but not basal cell density, played a protective role against conjunctivalisation (OR, 0.069; 95% CI 0.008–0.619; p=0.01). An SND cut-off value of 17 215 µm/mm2 yielded a sensitivity and specificity of 95.5% and 90.6%, respectively, for LSCD diagnosis. [Conclusion] The density of the corneal sub-basal nerve plexus was inversely related to conjunctivalisation in LSCD. Further studies are needed to verify this and to elucidate the directionality between these factors.

Inherited Retinal Dystrophies are clinically and genetically heterogeneous disorders affecting the photoreceptors. Although NGS has shown to be helpful for the molecular diagnosis of these conditions, some cases remain unsolved. Among these, several individuals harboured monoallelic variants in a recessive gene, suggesting that a comprehensive screening could improve the overall diagnosis. In order to assess the contribution of non-coding variations in a cohort of 29 patients, 25 of them with monoallelic mutations, we performed targeted NGS. The design comprised the entire genomic sequence of three genes (USH2A, ABCA4 and CEP290), the coding exons of 76 genes and two disease-associated intronic regions in OFD1 and PRPF31. As a result, likely causative mutations (8 novel) were identified in 17 probands (diagnostic rate: 58.62%), including two copy-number variations in USH2A (one deletion of exons 22–55 and one duplication of exons 46–47). Possibly damaging deep-intronic mutations were identified in one family, and another with a monoallelic variant harboured causal mutations in a different locus. In conclusion, due to the high prevalence of carriers of IRD mutations and the results obtained here, sequencing entire genes do not seem to be the approach of choice for detecting the second hit in IRD patients with monoallelic variants.

Retinitis Pigmentosa (RP) is the most common form of inherited retinal dystrophy (IRD) characterized ultimately by photoreceptors degeneration. Exhibiting great clinical and genetic heterogeneity, RP can be inherited as an autosomal dominant (ad), autosomal recessive (ar) and X-linked (xl) disorder. Although the relative prevalence of each form varies somewhat between populations, a major proportion (41% in Spain) of patients represent simplex cases (sRP) in which the mode of inheritance is unknown. Molecular genetic diagnostic is crucial, but also challenging, for sRP patients because any of the 81 RP genes identified to date may be causative. Herein, we report the use of a customized targeted gene panel consisting of 68 IRD genes for the molecular characterization of 106 sRP cases. The diagnostic rate was 62.26% (66 of 106) with a proportion of clinical refinements of 30.3%, demonstrating the high efficiency of this genomic approach even for clinically ambiguous cases. The high number of patients diagnosed here has allowed us to study in detail the genetic basis of the sRP. The solved sRP cohort is composed of 62.1% of arRP cases, 24.2% of adRP and 13.6% of xlRP, which implies consequences for counselling of patients and families.

Retinitis Pigmentosa (RP) is the most common form of inherited retinal dystrophy (IRD) characterized ultimately by photoreceptors degeneration. Exhibiting great clinical and genetic heterogeneity, RP can be inherited as an autosomal dominant (ad), autosomal recessive (ar) and X-linked (xl) disorder. Although the relative prevalence of each form varies somewhat between populations, a major proportion (41% in Spain) of patients represent simplex cases (sRP) in which the mode of inheritance is unknown. Molecular genetic diagnostic is crucial, but also challenging, for sRP patients because any of the 81 RP genes identified to date may be causative. Herein, we report the use of a customized targeted gene panel consisting of 68 IRD genes for the molecular characterization of 106 sRP cases. The diagnostic rate was 62.26% (66 of 106) with a proportion of clinical refinements of 30.3%, demonstrating the high efficiency of this genomic approach even for clinically ambiguous cases. The high number of patients diagnosed here has allowed us to study in detail the genetic basis of the sRP. The solved sRP cohort is composed of 62.1% of arRP cases, 24.2% of adRP and 13.6% of xlRP, which implies consequences for counselling of patients and families.

INTRODUCCIÓN La Insuficiencia limbar (IL) es una enfermedad ocular debida a una disminución crítica de las células madre limbares encargadas de la renovación del epitelio corneal y de constituir una barrera entre el epitelio conjuntival, opaco, y el corneal, translúcido. Es conocida la relación entre el epitelio corneal y los nervios del plexo nervioso subbasal de la córnea, existiendo un circuito químico retroalimentado para su correcto trofismo. La ausencia de epitelio corneal sano y la neovascularización de la superficie corneal se relacionan con la ausencia de plexo nervioso corneal de forma recíproca. Se ha relacionado la disminución de la densidad de nervios del plexo nervioso corneal subbasal o sub-epitelial (Subbasal nerve density, SND) y de la densidad de células basales (Basal cell density, BCD) con estadios precoces de IL. HIPÓTESIS El análisis de la densidad celular y del plexo nervioso subbasal del epitelio corneal mediante la microscopía confocal ayuda al diagnóstico precoz y a clasificar la insuficiencia limbar basándonos en pruebas documentables y de forma no invasiva. MATERIAL Y MÉTODOS En nuestro trabajo, hemos analizado 97 ojos de 49 pacientes, de los cuales, 85 ojos estaban diagnosticados clínicamente de IL y el resto actuaron de control. Se les realizó exploración oftalmológica completa, incluyendo fotografías de polo anterior, tinciones vitales, escala de Oxford y Van Bijsterveld, test de OSDI y VFQ 25, análisis con Pentacam®, Keratograph 5M® (K5M) y microscopio confocal HRT II con módulo de córnea Rostock®. Se catalogó a cada paciente según las clasificaciones de Rama, López-García, Deng clínica, Deng confocal y Schwartz. Para el análisis estadístico se ha usado paquete estadístico SPSS 22 y para el tamaño muestral el nQuery Advisor Release 7. Para relacionar dos variables cualitativas se realizaron tablas de contingencia y luego, prueba Chi-Cuadrado o bien, los métodos no asintóticos de Montecarlo y prueba Exacta. Para la comparación de varia, INTRODUCTION Limbal stem cell deficiency (LSCD) is an eye disease caused by a critical decrease in limbal stem cells responsible for the renewal of the corneal epithelium and form a barrier to the conjunctival epithelium, opaque and vascularized epithelium. It is known the relationship between the corneal epithelium and neural subasal corneal nerve plexus, having a feedback circuit. The lack of healthy corneal epithelium and corneal neovascularization surface relates to the absence of corneal nerve plexus reciprocally. It has been associated with decreased density of the corneal nerve plexus nerves (SND) and basal cell density (BCD) with early stages of IL. HYPOTHESIS Analysis of cell density and subasal corneal epithelial nerve plexus using confocal microscopy help to early diagnosis and classify LSCD based on documentable and non-invasive tests. MATERIAL AND METHODS In our work, we have analyzed 97 eyes of 49 patients, of whom 85 eyes were diagnosed clinically and the rest of IL acted as controls. They underwent complete eye examination, including photographs of the anterior pole, vital stains, Oxford and Van Bijsterveld scales, OSDI and VFQ 25 questionnaires, Pentacam® tomography, Keratograph 5M® (K5M) and HRT II confocal microscopy with Rostock® cornea module. We classified these patients through Rama, Lopez-Garcia, Deng clinic, confocal Deng and Schwartz classifications. SPSS 22 was used for statistical analysis and nQuery Advisor Release 7 for sample size. To make the ordered logistic regression model, multivariate analysis was done by a non-automated method of direct introduction of variables, measuring the reliability with Hosmer-Lemeshow index. Agreement of classifications was measured through Cohen's kappa coefficient. RESULTS SND was analyzed with confocal microscopy in patients with IL: 6469 (6295) microns / mm2; lower than our healthy controls, 21766 (4970) microns / mm2) and reference numbers described in the literature,19000 microns/ mm2 (p <0.0005).

Next-generation sequencing (NGS) has overcome important limitations to the molecular diagnosis of Inherited Retinal Dystrophies (IRD) such as the high clinical and genetic heterogeneity and the overlapping phenotypes. The purpose of this study was the identification of the genetic defect in 32 Spanish families with different forms of IRD. With that aim, we implemented a custom NGS panel comprising 64 IRD-associated genes in our population, and three disease-associated intronic regions. A total of 37 pathogenic mutations (14 novels) were found in 73% of IRD patients ranging from 50% for autosomal dominant cases, 75% for syndromic cases, 83% for autosomal recessive cases, and 100% for X-linked cases. Additionally, unexpected phenotype-genotype correlations were found in 6 probands, which led to the refinement of their clinical diagnoses. Furthermore, intra- and interfamilial phenotypic variability was observed in two cases. Moreover, two cases unsuccessfully analysed by exome sequencing were resolved by applying this panel. Our results demonstrate that this hypothesis-free approach based on frequently mutated, population-specific loci is highly cost-efficient for the routine diagnosis of this heterogeneous condition and allows the unbiased analysis of a miscellaneous cohort. The molecular information found here has aid clinical diagnosis and has improved genetic counselling and patient management.

Next-generation sequencing (NGS) has overcome important limitations to the molecular diagnosis of Inherited Retinal Dystrophies (IRD) such as the high clinical and genetic heterogeneity and the overlapping phenotypes. The purpose of this study was the identification of the genetic defect in 32 Spanish families with different forms of IRD. With that aim, we implemented a custom NGS panel comprising 64 IRD-associated genes in our population, and three disease-associated intronic regions. A total of 37 pathogenic mutations (14 novels) were found in 73% of IRD patients ranging from 50% for autosomal dominant cases, 75% for syndromic cases, 83% for autosomal recessive cases, and 100% for X-linked cases. Additionally, unexpected phenotype-genotype correlations were found in 6 probands, which led to the refinement of their clinical diagnoses. Furthermore, intra- and interfamilial phenotypic variability was observed in two cases. Moreover, two cases unsuccessfully analysed by exome sequencing were resolved by applying this panel. Our results demonstrate that this hypothesis-free approach based on frequently mutated, population-specific loci is highly cost-efficient for the routine diagnosis of this heterogeneous condition and allows the unbiased analysis of a miscellaneous cohort. The molecular information found here has aid clinical diagnosis and has improved genetic counselling and patient management.