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1.
Int J Mol Sci ; 25(5)2024 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-38474159

RESUMO

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.


Assuntos
Distrofias Retinianas , Retinose Pigmentar , Humanos , Análise Mutacional de DNA , Mutação , Mutação de Sentido Incorreto , Fenótipo , Distrofias Retinianas/genética , Retinose Pigmentar/genética
2.
Clin Genet ; 103(2): 236-241, 2023 02.
Artigo em Inglês | MEDLINE | ID: mdl-36250766

RESUMO

The biallelic pathogenic repeat (AAGGG)400-2000 intronic expansion in the RFC1 gene has been recently described as the cause of cerebellar ataxia, neuropathy, vestibular areflexia syndrome (CANVAS) and as a major cause of late-onset ataxia. Since then, many heterozygous carriers have been identified, with an estimated allele frequency of 0.7% to 4% in the healthy population. Here, we describe in two affected CANVAS sisters the presence of the nonsense c.724C > T p.(Arg242*) variant in compound heterozygosity with the pathogenic repeat expansion in the RFC1 gene. Further RNA analysis demonstrated a reduced expression of the p.Arg242* allele in patients confirming an efficient nonsense-mediated mRNA decay. We also highlight the importance of considering the sequencing of the RFC1 gene for the diagnosis, especially in patients with CANVAS diagnosis carriers of the AAGGG repeat expansion.


Assuntos
Vestibulopatia Bilateral , Ataxia Cerebelar , Doenças do Sistema Nervoso Periférico , Proteína de Replicação C , Neuronite Vestibular , Humanos , Ataxia/genética , Vestibulopatia Bilateral/genética , Ataxia Cerebelar/genética , Ataxia Cerebelar/diagnóstico , Doenças do Sistema Nervoso Periférico/diagnóstico , Doenças do Sistema Nervoso Periférico/genética , Síndrome , Doenças Vestibulares/genética , Neuronite Vestibular/genética , Proteína de Replicação C/genética
3.
Nucleic Acids Res ; 49(D1): D1130-D1137, 2021 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-32990755

RESUMO

The knowledge of the genetic variability of the local population is of utmost importance in personalized medicine and has been revealed as a critical factor for the discovery of new disease variants. Here, we present the Collaborative Spanish Variability Server (CSVS), which currently contains more than 2000 genomes and exomes of unrelated Spanish individuals. This database has been generated in a collaborative crowdsourcing effort collecting sequencing data produced by local genomic projects and for other purposes. Sequences have been grouped by ICD10 upper categories. A web interface allows querying the database removing one or more ICD10 categories. In this way, aggregated counts of allele frequencies of the pseudo-control Spanish population can be obtained for diseases belonging to the category removed. Interestingly, in addition to pseudo-control studies, some population studies can be made, as, for example, prevalence of pharmacogenomic variants, etc. In addition, this genomic data has been used to define the first Spanish Genome Reference Panel (SGRP1.0) for imputation. This is the first local repository of variability entirely produced by a crowdsourcing effort and constitutes an example for future initiatives to characterize local variability worldwide. CSVS is also part of the GA4GH Beacon network. CSVS can be accessed at: http://csvs.babelomics.org/.


Assuntos
Crowdsourcing , Bases de Dados Genéticas , Genética Populacional/métodos , Genoma Humano , Software , Alelos , Mapeamento Cromossômico , Exoma , Frequência do Gene , Variação Genética , Genômica , Humanos , Internet , Medicina de Precisão/métodos , Espanha
4.
Mol Vis ; 28: 48-56, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35693422

RESUMO

Purpose: To describe the clinical phenotype and genetic basis of non-syndromic retinitis pigmentosa (RP) in one family and two sporadic cases with biallelic mutations in the transcription factor neural retina leucine zipper (NRL). Methods: Exome sequencing was performed in one affected family member. Microsatellite genotyping was used for haplotype analysis. PCR and Sanger sequencing were used to confirm mutations in and screen other family members where they were available. The SMART tool for domain prediction helped us build the protein schematic diagram. Results: For family MM1 of Pakistani origin, whole-exome sequencing and microsatellite genotyping revealed homozygosity on chromosome 14 and identified a homozygous stop-loss mutation in NRL, NM_006177.5: c.713G>T, p.*238Lext57, which is predicted to add an extra 57 amino acids to the normal protein chain. The variant segregated with disease symptoms in the family. For case RP-3051 of Spanish ancestry, clinical exome sequencing focusing on the morbid genome highlighted a homozygous nonsense mutation in NRL, c.238C>T, p.Gln80*, as the most likely disease candidate. For case RP-1553 of Romanian ethnicity, targeted-exome sequencing of 73 RP/LCA genes identified a homozygous nonsense mutation in NRL, c.544C>T, p.Gln182*. The variants were either rare or absent in the gnomAD database. Conclusions: NRL mutations predominantly cause dominant retinal disease, but there have been five published reports of mutations causing recessive disease. Here, we present three further examples of recessive RP due to NRL mutations. The phenotypes observed are consistent with those in the previous reports, and the observed mutation types and distribution further confirm distinct patterns for variants in NRL causing recessive and dominant diseases.


Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/genética , Proteínas do Olho/genética , Retinose Pigmentar , Fatores de Transcrição , Códon sem Sentido , Análise Mutacional de DNA , Humanos , Mutação , Linhagem , Retinose Pigmentar/diagnóstico , Retinose Pigmentar/genética , Fatores de Transcrição/genética
5.
J Med Genet ; 58(8): 570-578, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-32817297

RESUMO

BACKGROUND: Inherited retinal disorders are a clinically and genetically heterogeneous group of conditions and a major cause of visual impairment. Common disease subtypes include vitelliform macular dystrophy (VMD) and retinitis pigmentosa (RP). Despite the identification of over 90 genes associated with RP, conventional genetic testing fails to detect a molecular diagnosis in about one third of patients with RP. METHODS: Exome sequencing was carried out for identifying the disease-causing gene in a family with autosomal dominant RP. Gene panel testing and exome sequencing were performed in 596 RP and VMD families to identified additional IMPG1 variants. In vivo analysis in the medaka fish system by knockdown assays was performed to screen IMPG1 possible pathogenic role. RESULTS: Exome sequencing of a family with RP revealed a splice variant in IMPG1. Subsequently, the same variant was identified in individuals from two families with either RP or VMD. A retrospective study of patients with RP or VMD revealed eight additional families with different missense or nonsense variants in IMPG1. In addition, the clinical diagnosis of the IMPG1 retinopathy-associated variant, originally described as benign concentric annular macular dystrophy, was also revised to RP with early macular involvement. Using morpholino-mediated ablation of Impg1 and its paralog Impg2 in medaka fish, we confirmed a phenotype consistent with that observed in the families, including a decreased length of rod and cone photoreceptor outer segments. CONCLUSION: This study discusses a previously unreported association between monoallelic or biallelic IMPG1 variants and RP. Notably, similar observations have been reported for IMPG2.


Assuntos
Proteínas da Matriz Extracelular , Proteínas do Olho , Genes Recessivos , Predisposição Genética para Doença , Mutação , Proteoglicanas , Retinose Pigmentar , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Exoma/genética , Sequenciamento do Exoma/métodos , Proteínas da Matriz Extracelular/genética , Proteínas do Olho/genética , Genes Recessivos/genética , Predisposição Genética para Doença/genética , Padrões de Herança/genética , Degeneração Macular/genética , Mutação/genética , Linhagem , Fenótipo , Proteoglicanas/genética , Retina/patologia , Retinose Pigmentar/genética , Estudos Retrospectivos
6.
Int J Mol Sci ; 23(15)2022 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-35955564

RESUMO

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.


Assuntos
Distrofias Retinianas , Estudos de Coortes , Genômica , Humanos , Mutação , Linhagem , Distrofias Retinianas/diagnóstico , Distrofias Retinianas/genética , Estudos Retrospectivos , Sequenciamento do Exoma
7.
Exp Eye Res ; 212: 108761, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34492281

RESUMO

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.


Assuntos
DNA/genética , Estudos de Associação Genética/métodos , Mutação , Distrofias Retinianas/genética , cis-trans-Isomerases/genética , Adolescente , Alelos , Criança , Pré-Escolar , Análise Mutacional de DNA , Eletrorretinografia , Feminino , Genótipo , Humanos , Lactente , Masculino , Linhagem , Fenótipo , Distrofias Retinianas/diagnóstico , Distrofias Retinianas/metabolismo , Adulto Jovem , cis-trans-Isomerases/metabolismo
8.
Int J Mol Sci ; 22(4)2021 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-33672445

RESUMO

Hereditary retinal dystrophies (HRD) represent a significant cause of blindness, affecting mostly retinal pigment epithelium (RPE) and photoreceptors (PRs), and currently suffer from a lack of effective treatments. Highly specialized RPE and PR cells interact mutually in the functional retina, therefore primary HRD affecting one cell type leading to a secondary HRD in the other cells. Phagocytosis is one of the primary functions of the RPE and studies have discovered that mutations in the phagocytosis-associated gene Mer tyrosine kinase receptor (MERTK) lead to primary RPE dystrophy. Treatment strategies for this rare disease include the replacement of diseased RPE with healthy autologous RPE to prevent PR degeneration. The generation and directed differentiation of patient-derived human-induced pluripotent stem cells (hiPSCs) may provide a means to generate autologous therapeutically-relevant adult cells, including RPE and PR. However, the continued presence of the MERTK gene mutation in patient-derived hiPSCs represents a significant drawback. Recently, we reported the generation of a hiPSC model of MERTK-associated Retinitis Pigmentosa (RP) that recapitulates disease phenotype and the subsequent creation of gene-corrected RP-hiPSCs using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9. In this study, we differentiated gene-corrected RP-hiPSCs into RPE and found that these cells had recovered both wild-type MERTK protein expression and the lost phagocytosis of fluorescently-labeled photoreceptor outer segments observed in uncorrected RP-hiPSC-RPE. These findings provide proof-of-principle for the utility of gene-corrected hiPSCs as an unlimited cell source for personalized cell therapy of rare vision disorders.


Assuntos
Edição de Genes , Células-Tronco Pluripotentes Induzidas/patologia , Fagocitose , Epitélio Pigmentado da Retina/patologia , Retinose Pigmentar/patologia , Diferenciação Celular/genética , Linhagem Celular , Regulação da Expressão Gênica , Humanos , Células-Tronco Pluripotentes Induzidas/ultraestrutura , Mutação/genética , Segmento Externo das Células Fotorreceptoras da Retina/metabolismo , Segmento Externo das Células Fotorreceptoras da Retina/patologia , Segmento Externo das Células Fotorreceptoras da Retina/ultraestrutura , Epitélio Pigmentado da Retina/ultraestrutura , Retinose Pigmentar/genética , c-Mer Tirosina Quinase/genética , c-Mer Tirosina Quinase/metabolismo
9.
Mol Vis ; 26: 216-225, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32214787

RESUMO

Purpose: The aim of the present work is the molecular diagnosis of three patients with deafness and retinal degeneration. Methods: Three patients from two unrelated families were initially analyzed with custom gene panels for Usher genes, non-syndromic hearing loss, or inherited syndromic retinopathies and further investigated by means of clinical or whole exome sequencing. Results: The study allowed us to detect likely pathogenic variants in PEX6, a gene typically involved in peroxisomal biogenesis disorders (PBDs). Beside deaf-blindness, both families showed additional features: Siblings from Family 1 showed enamel alteration and abnormal peroxisome. In addition, the brother had mild neurodevelopmental delay and nephrolithiasis. The case II:1 from Family 2 showed intellectual disability, enamel alteration, and dysmorphism. Conclusions: We have reported three new cases with pathogenic variants in PEX6 presenting with milder forms of the Zellweger spectrum disorders (ZSD). The three cases showed distinct clinical features. Thus, expanding the phenotypic spectrum of PBDs and ascertaining exome sequencing is an effective strategy for an accurate diagnosis of clinically overlapping and genetically heterogeneous disorders such as deafness-blindness association.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/genética , Perda Auditiva Neurossensorial/genética , Retinose Pigmentar/genética , Síndrome de Zellweger/genética , Adulto , Criança , Anormalidades Craniofaciais/genética , Esmalte Dentário/anormalidades , Feminino , Humanos , Deficiência Intelectual/genética , Masculino , Mutação , Nefrolitíase/genética , Transtornos do Neurodesenvolvimento/genética , Linhagem , Peroxissomos/genética , Peroxissomos/metabolismo , Peroxissomos/patologia , Sequenciamento do Exoma
10.
Ophthalmology ; 126(8): 1181-1188, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-30902645

RESUMO

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.


Assuntos
Retinose Pigmentar/genética , Adulto , Estudos de Coortes , Hibridização Genômica Comparativa , Variações do Número de Cópias de DNA , Análise Mutacional de DNA/métodos , Feminino , Genes Recessivos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Linhagem , Fenótipo
11.
Hum Mol Genet ; 24(14): 4037-48, 2015 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-25882705

RESUMO

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.


Assuntos
Proteínas de Ligação a DNA/genética , Exoma , Estudo de Associação Genômica Ampla , Retinose Pigmentar/genética , Fatores de Transcrição/genética , Sequência de Aminoácidos , Animais , Células COS , Chlorocebus aethiops , Mapeamento Cromossômico , Proteínas de Ligação a DNA/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Homozigoto , Humanos , Dados de Sequência Molecular , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Linhagem , Retina/patologia , Células Fotorreceptoras Retinianas Cones/patologia , Células Fotorreceptoras Retinianas Bastonetes/patologia , Fatores de Transcrição/metabolismo
12.
Am J Hum Genet ; 93(3): 571-8, 2013 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-23993198

RESUMO

Vitelliform macular dystrophies (VMD) are inherited retinal dystrophies characterized by yellow, round deposits visible upon fundus examination and encountered in individuals with juvenile Best macular dystrophy (BMD) or adult-onset vitelliform macular dystrophy (AVMD). Although many BMD and some AVMD cases harbor mutations in BEST1 or PRPH2, the underlying genetic cause remains unknown for many affected individuals. In a large family with autosomal-dominant VMD, gene mapping and whole-exome sequencing led to the identification of a c.713T>G (p.Leu238Arg) IMPG1 mutation, which was subsequently found in two other families with autosomal-dominant VMD and the same phenotype. IMPG1 encodes the SPACR protein, a component of the rod and cone photoreceptor extracellular matrix domains. Structural modeling indicates that the p.Leu238Arg substitution destabilizes the conserved SEA1 domain of SPACR. Screening of 144 probands who had various forms of macular dystrophy revealed three other IMPG1 mutations. Two individuals from one family affected by autosomal-recessive VMD were homozygous for the splice-site mutation c.807+1G>T, and two from another family were compound heterozygous for the mutations c.461T>C (p.Leu154Pro) and c.1519C>T (p.Arg507(∗)). Most cases had a normal or moderately decreased electrooculogram Arden ratio. We conclude that IMPG1 mutations cause both autosomal-dominant and -recessive forms of VMD, thus indicating that impairment of the interphotoreceptor matrix might be a general cause of VMD.


Assuntos
Proteínas da Matriz Extracelular/genética , Proteínas do Olho/genética , Predisposição Genética para Doença , Mutação/genética , Proteoglicanas/genética , Distrofia Macular Viteliforme/genética , Adulto , Sequência de Aminoácidos , Sequência de Bases , Cromossomos Humanos/genética , Proteínas da Matriz Extracelular/química , Proteínas do Olho/química , Feminino , Fundo de Olho , Humanos , Padrões de Herança/genética , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Linhagem , Fenótipo , Proteoglicanas/química , Adulto Jovem
13.
Mol Vis ; 20: 843-51, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24959063

RESUMO

PURPOSE: Mutations in genes encoding proteins from the tri-snRNP complex of the spliceosome account for more than 12% of cases of autosomal dominant retinitis pigmentosa (adRP). Although the exact mechanism by which splicing factor defects trigger photoreceptor death is not completely clear, their role in retinitis pigmentosa has been demonstrated by several genetic and functional studies. To test for possible novel associations between splicing factors and adRP, we screened four tri-snRNP splicing factor genes (EFTUD2, PRPF4, NHP2L1, and AAR2) as candidate disease genes. METHODS: We screened up to 303 patients with adRP from Europe and North America who did not carry known RP mutations. Exon-PCR and Sanger methods were used to sequence the NHP2L1 and AAR2 genes, while the sequences of EFTUD2 and PRPF4 were obtained by using long-range PCRs spanning coding and non-coding regions followed by next-generation sequencing. RESULTS: We detected novel missense changes in individual patients in the sequence of the genes PRPF4 and EFTUD2, but the role of these changes in relationship to disease could not be verified. In one other patient we identified a novel nucleotide substitution in the 5' untranslated region (UTR) of NHP2L1, which did not segregate with the disease in the family. CONCLUSIONS: The absence of clearly pathogenic mutations in the candidate genes screened in our cohort suggests that EFTUD2, PRPF4, NHP2L1, and AAR2 are either not involved in adRP or are associated with the disease in rare instances, at least as observed in this study in patients of European and North American origin.


Assuntos
Análise Mutacional de DNA/métodos , Genes Dominantes , Testes Genéticos , Splicing de RNA/genética , Retinose Pigmentar/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Fases de Leitura Aberta/genética , Fatores de Alongamento de Peptídeos/genética , Ribonucleoproteína Nuclear Pequena U4-U6/genética , Ribonucleoproteína Nuclear Pequena U5 , Ribonucleoproteínas Nucleares Pequenas/genética
14.
Ophthalmology ; 121(1): 399-407, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24144451

RESUMO

OBJECTIVE: We aimed to identify novel genetic defects in the LCA5 gene underlying Leber congenital amaurosis (LCA) in the Spanish population and to describe the associated phenotype. DESIGN: Case series. PARTICIPANTS: A cohort of 217 unrelated Spanish families affected by autosomal recessive or isolated retinal dystrophy, that is, 79 families with LCA and 138 families with early-onset retinitis pigmentosa (EORP). A total of 100 healthy, unrelated Spanish individuals were screened as controls. METHODS: High-resolution homozygosity mapping was performed in 44 patients with LCA using genome-wide single nucleotide polymorphism (SNP) microarrays. Direct sequencing of the LCA5 gene was performed in 5 patients who showed homozygous regions at chromosome 6 and in 173 unrelated individuals with LCA or EORP. The ophthalmic history of 8 patients carrying LCA5 mutations was reviewed and additional examinations were performed, including electroretinography (ERG), optical coherence tomography (OCT), and fundus photography. MAIN OUTCOME MEASURES: Single nucleotide polymorphism genotyping, identity-by-descent (IBD) regions, LCA5 mutations, best-corrected visual acuity, visual field assessments, fundus appearance, ERG, and OCT findings. RESULTS: Four novel and 2 previously reported LCA5 mutations have been identified in 6 unrelated families with LCA by homozygosity mapping or Sanger sequencing. Thus, LCA5 mutations have a frequency of 7.6% in the Spanish population. However, no LCA5 mutations were found in 138 patients with EORP. Although most of the identified LCA5 mutations led to a truncated protein, a likely pathogenic missense variant was identified for the first time as a cause of LCA, segregating in 2 families. We also have characterized a novel splicing site mutation at the RNA level, demonstrating that the mutant LCA5 transcript was absent in a patient. All patients carrying LCA5 mutations presented nystagmus, night blindness, and progressive loss of visual acuity and visual field leading to blindness toward the third decade of life. Fundoscopy showed fundus features of pigmentary retinopathy with atrophic macular lesions. CONCLUSIONS: This work reveals a higher frequency of LCA5 mutations in a Spanish LCA cohort than in other populations. This study established gene-specific frequencies and the underlying phenotype of LCA5 mutations in the Spanish population.


Assuntos
Proteínas do Olho/genética , Amaurose Congênita de Leber/genética , Proteínas Associadas aos Microtúbulos/genética , Mutação de Sentido Incorreto , Polimorfismo de Nucleotídeo Único , Retinose Pigmentar/genética , Adulto , Criança , Cromossomos Humanos Par 6/genética , Eletrorretinografia , Frequência do Gene , Estudo de Associação Genômica Ampla , Técnicas de Genotipagem , Humanos , Análise em Microsséries , Fenótipo , Polimorfismo de Nucleotídeo Único/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espanha , Acuidade Visual , Campos Visuais , Adulto Jovem
15.
Ophthalmology ; 121(8): 1620-7, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24697911

RESUMO

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.


Assuntos
Ataxia/genética , Catarata/genética , Exoma/genética , Monoacilglicerol Lipases/genética , Mutação de Sentido Incorreto , Polineuropatias/genética , Retinose Pigmentar/genética , Adulto , Idoso , Ataxia/diagnóstico , Ataxia/fisiopatologia , Audiometria , Catarata/diagnóstico , Catarata/fisiopatologia , Eletrorretinografia , Feminino , Genes Recessivos , Humanos , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Monoacilglicerol Lipases/química , Linhagem , Fenótipo , Polineuropatias/diagnóstico , Polineuropatias/fisiopatologia , Estrutura Secundária de Proteína , Retinose Pigmentar/diagnóstico , Retinose Pigmentar/fisiopatologia , Análise de Sequência de DNA , Acuidade Visual/fisiologia , Campos Visuais/fisiologia
16.
Am J Hum Genet ; 86(5): 686-95, 2010 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-20398886

RESUMO

Retinitis pigmentosa is a genetically heterogeneous group of inherited ocular disorders characterized by progressive photoreceptor cell loss, night blindness, constriction of the visual field, and progressive visual disability. Homozygosity mapping and gene expression studies identified a 2 exon gene, C2ORF71. The encoded protein has no homologs and is highly expressed in the eye, where it is specifically expressed in photoreceptor cells. Two mutations were found in C2ORF71 in human RP patients: A nonsense mutation (p.W253X) in the first exon is likely to be a null allele; the second, a missense mutation (p.I201F) within a highly conserved region of the protein, leads to proteosomal degradation. Bioinformatic and functional studies identified and validated sites of lipid modification within the first three amino acids of the C2ORF71 protein. Using morpholino oligonucleotides to knockdown c2orf71 expression in zebrafish results in visual defects, confirming that C2ORF71 plays an important role in the development of normal vision. Finally, localization of C2ORF71 to primary cilia in cultured cells suggests that the protein is likely to localize to the connecting cilium or outer segment of photoreceptor cells.


Assuntos
Olho/metabolismo , Mutação , Células Fotorreceptoras de Vertebrados/metabolismo , Proteínas/genética , Retinose Pigmentar/genética , Cegueira/genética , Cílios/genética , Cílios/metabolismo , Éxons , Proteínas do Olho/genética , Homozigoto , Humanos , Mutação de Sentido Incorreto , Retinose Pigmentar/metabolismo
17.
Ophthalmology ; 120(11): 2332-7, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23755871

RESUMO

OBJECTIVE: To provide a comprehensive overview of all detected mutations in the ABCA4 gene in Spanish families with autosomal recessive retinal disorders, including Stargardt's disease (arSTGD), cone-rod dystrophy (arCRD), and retinitis pigmentosa (arRP), and to assess genotype-phenotype correlation and disease progression in 10 years by considering the type of variants and age at onset. DESIGN: Case series. PARTICIPANTS: A total of 420 unrelated Spanish families: 259 arSTGD, 86 arCRD, and 75 arRP. METHODS: Spanish families were analyzed through a combination of ABCR400 genotyping microarray, denaturing high-performance liquid chromatography, and high-resolution melting scanning. Direct sequencing was used as a confirmation technique for the identified variants. Screening by multiple ligation probe analysis was used to detect possible large deletions or insertions in the ABCA4 gene. Selected families were analyzed further by next generation sequencing. MAIN OUTCOME MEASURES: DNA sequence variants, mutation detection rates, haplotypes, age at onset, central or peripheral vision loss, and night blindness. RESULTS: Overall, we detected 70.5% and 36.6% of all expected ABCA4 mutations in arSTGD and arCRD patient cohorts, respectively. In the fraction of the cohort where the ABCA4 gene was sequenced completely, the detection rates reached 73.6% for arSTGD and 66.7% for arCRD. However, the frequency of possibly pathogenic ABCA4 alleles in arRP families was only slightly higher than that in the general population. Moreover, in some families, mutations in other known arRP genes segregated with the disease phenotype. CONCLUSIONS: An increasing understanding of causal ABCA4 alleles in arSTGD and arCRD facilitates disease diagnosis and prognosis and also is paramount in selecting patients for emerging clinical trials of therapeutic interventions. Because ABCA4-associated diseases are evolving retinal dystrophies, assessment of age at onset, accurate clinical diagnosis, and genetic testing are crucial. We suggest that ABCA4 mutations may be associated with a retinitis pigmentosa-like phenotype often as a consequence of severe (null) mutations, in cases of long-term, advanced disease, or both. Patients with classical arRP phenotypes, especially from the onset of the disease, should be screened first for mutations in known arRP genes and not ABCA4.


Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Mutação , Retinose Pigmentar/genética , Adolescente , Adulto , Idade de Início , Alelos , Criança , Pré-Escolar , Cromatografia Líquida de Alta Pressão , Eletrorretinografia , Angiofluoresceinografia , Estudos de Associação Genética , Técnicas de Genotipagem , Humanos , Degeneração Macular/diagnóstico , Degeneração Macular/genética , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase Multiplex , Retinose Pigmentar/diagnóstico , Estudos Retrospectivos , Espanha , Doença de Stargardt , Adulto Jovem
18.
Front Genet ; 14: 1234032, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37779911

RESUMO

Introduction: Inherited retinal dystrophies (IRDs) can be caused by variants in more than 280 genes. The ATP-binding cassette transporter type A4 (ABCA4) gene is one of these genes and has been linked to Stargardt disease type 1 (STGD1), fundus flavimaculatus, cone-rod dystrophy (CRD), and pan-retinal CRD. Approximately 25% of the reported ABCA4 variants affect RNA splicing. In most cases, it is necessary to perform a functional assay to determine the effect of these variants. Methods: Whole genome sequencing (WGS) was performed in one Spanish proband with Stargardt disease. The putative pathogenicity of c.6480-35A>G on splicing was investigated both in silico and in vitro. The in silico approach was based on the deep-learning tool SpliceAI. For the in vitro approach we used a midigene splice assay in HEK293T cells, based on a previously established wild-type midigene (BA29) containing ABCA4 exons 46 to 48. Results: Through the analysis of WGS data, we identified two candidate variants in ABCA4 in one proband: a previously described deletion, c.699_768+342del (p.(Gln234Phefs*5)), and a novel branchpoint variant, c.6480-35A>G. Segregation analysis confirmed that the variants were in trans. For the branchpoint variant, SpliceAI predicted an acceptor gain with a high score (0.47) at position c.6480-47. A midigene splice assay in HEK293T cells revealed the inclusion of the last 47 nucleotides of intron 47 creating a premature stop codon and allowed to categorize the variant as moderately severe. Subsequent analysis revealed the presence of this variant as a second allele besides c.1958G>A p.(Arg653His) in an additional Spanish proband in a large cohort of IRD cases. Conclusion: A splice-altering effect of the branchpoint variant, confirmed by the midigene splice assay, along with the identification of this variant in a second unrelated individual affected with STGD, provides sufficient evidence to classify the variant as likely pathogenic. In addition, this research highlights the importance of studying non-coding regions and performing functional assays to provide a conclusive molecular diagnosis.

19.
Am J Ophthalmol ; 254: 87-103, 2023 10.
Artigo em Inglês | MEDLINE | ID: mdl-37327959

RESUMO

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.


Assuntos
Distrofias de Cones e Bastonetes , Amaurose Congênita de Leber , Cegueira Noturna , Humanos , Distrofias de Cones e Bastonetes/diagnóstico , Distrofias de Cones e Bastonetes/genética , Genótipo , Amaurose Congênita de Leber/diagnóstico , Amaurose Congênita de Leber/genética , Mutação , Cegueira Noturna/diagnóstico , Cegueira Noturna/genética , Linhagem , Fenótipo , Estudos Retrospectivos
20.
Br J Ophthalmol ; 2023 Oct 18.
Artigo em Inglês | MEDLINE | ID: mdl-37852740

RESUMO

BACKGROUND/AIMS: To investigate genotype-phenotype associations in patients with KCNV2 retinopathy. METHODS: Review of clinical notes, best-corrected visual acuity (BCVA), molecular variants, electroretinography (ERG) and retinal imaging. Subjects were grouped according to the combination of KCNV2 variants-two loss-of-function (TLOF), two missense (TM) or one of each (MLOF)-and parameters were compared. RESULTS: Ninety-two patients were included. The mean age of onset (mean±SD) in TLOF (n=55), TM (n=23) and MLOF (n=14) groups was 3.51±0.58, 4.07±2.76 and 5.54±3.38 years, respectively. The mean LogMAR BCVA (±SD) at baseline in TLOF, TM and MLOF groups was 0.89±0.25, 0.67±0.38 and 0.81±0.35 for right, and 0.88±0.26, 0.69±0.33 and 0.78±0.33 for left eyes, respectively. The difference in BCVA between groups at baseline was significant in right (p=0.03) and left eyes (p=0.035). Mean outer nuclear layer thickness (±SD) at baseline in TLOF, MLOF and TM groups was 37.07±15.20 µm, 40.67±12.53 and 40.38±18.67, respectively, which was not significantly different (p=0.85). The mean ellipsoid zone width (EZW) loss (±SD) was 2051 µm (±1318) for patients in the TLOF, and 1314 µm (±965) for MLOF. Only one patient in the TM group had EZW loss at presentation. There was considerable overlap in ERG findings, although the largest DA 10 ERG b-waves were associated with TLOF and the smallest with TM variants. CONCLUSIONS: Patients with missense alterations had better BCVA and greater structural integrity. This is important for patient prognostication and counselling, as well as stratification for future gene therapy trials.

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