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Purpose: We investigated the natural history of retinal dystrophy owing to variants in the MYO7A gene. Methods: Fifty-three patients (mean age, 33.6 ± 16.7 years) with Usher syndrome owing to biallelic, mostly pathogenic, variants in MYO7A underwent baseline and two annual follow-up visits. Best-corrected visual acuity (BCVA), semiautomatic kinetic visual field, full-field electroretinogram, color fundus imaging, microperimetry, spectral-domain optical coherence tomography, and fundus autofluorescence were assessed. Results: At baseline, all patients presented with decreased BCVA (66.4 ± 17.9 Early Treatment Diabetic Retinopathy score and 59.5 ± 21.7 Early Treatment Diabetic Retinopathy score, in the better- and worse-seeing eyes, respectively), restricted semiautomatic kinetic visual field (III4e area, 3365.8 ± 4142.1°2; 4176.4 ± 4400.3°2) and decreased macular sensitivity (9.7 ± 9.9 dB; 9.0 ± 10.2 dB). Spectral-domain optical coherence tomography revealed reduced central macular thickness (259.6 ± 63.0 µm; 250.7 ± 63.3 µm) and narrowed ellipsoid zone band width (2807.5 ± 2374.6 µm; 2615.5 ± 2370.4 µm). Longitudinal analyses (50 patients) showed a significant decrease of BCVA in better-seeing eyes, whereas no changes were observed in worse-seeing eyes for any parameter. BCVA, semiautomatic kinetic visual field (III4e and V4e) and macular sensitivity were related significantly to age at baseline. Hyperautofluorescent foveal patch (16 eyes [31.4%]) and abnormal central hypoautofluorescence (9 eyes [17.6%]) were significantly associated with worse morphological and functional read-outs compared with the hyperautofluorescent ring pattern (22 eyes [43.1%]). Conclusions: Our European multicentric study offers the first prospective longitudinal analysis in one of the largest cohorts of MYO7A patients described to date, confirming the slow disease progression. More important, this study emphasizes the key role of fundus autofluorescence patterns in retinal impairment staging and advocates its adoption as an objective biomarker in patient selection for future gene therapy clinical trials.
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Eletrorretinografia , Terapia Genética , Miosina VIIa , Tomografia de Coerência Óptica , Síndromes de Usher , Acuidade Visual , Campos Visuais , Humanos , Masculino , Feminino , Adulto , Estudos Prospectivos , Tomografia de Coerência Óptica/métodos , Acuidade Visual/fisiologia , Pessoa de Meia-Idade , Campos Visuais/fisiologia , Adulto Jovem , Adolescente , Síndromes de Usher/genética , Síndromes de Usher/fisiopatologia , Síndromes de Usher/terapia , Síndromes de Usher/diagnóstico , Terapia Genética/métodos , Criança , Testes de Campo Visual , Europa (Continente) , Angiofluoresceinografia , Seguimentos , Idoso , Estudos Longitudinais , Progressão da Doença , Miosinas/genética , Retina/diagnóstico por imagem , Retina/fisiopatologia , Retina/patologiaRESUMO
ABCA4 is the most frequently mutated gene leading to inherited retinal disease (IRD) with over 2200 pathogenic variants reported to date. Of these, ~1% are copy number variants (CNVs) involving the deletion or duplication of genomic regions, typically >50 nucleotides in length. An in-depth assessment of the current literature based on the public database LOVD, regarding the presence of known CNVs and structural variants in ABCA4, and additional sequencing analysis of ABCA4 using single-molecule Molecular Inversion Probes (smMIPs) for 148 probands highlighted recurrent and novel CNVs associated with ABCA4-associated retinopathies. An analysis of the coverage depth in the sequencing data led to the identification of eleven deletions (six novel and five recurrent), three duplications (one novel and two recurrent) and one complex CNV. Of particular interest was the identification of a complex defect, i.e., a 15.3 kb duplicated segment encompassing exon 31 through intron 41 that was inserted at the junction of a downstream 2.7 kb deletion encompassing intron 44 through intron 47. In addition, we identified a 7.0 kb tandem duplication of intron 1 in three cases. The identification of CNVs in ABCA4 can provide patients and their families with a genetic diagnosis whilst expanding our understanding of the complexity of diseases caused by ABCA4 variants.
Assuntos
Transportadores de Cassetes de Ligação de ATP , Variações do Número de Cópias de DNA , Doenças Retinianas , Humanos , Transportadores de Cassetes de Ligação de ATP/genética , Doenças Retinianas/genética , Feminino , Masculino , Linhagem , Íntrons/genética , Éxons/genética , Duplicação GênicaRESUMO
Inherited retinal diseases (IRDs) are a group of rare monogenic diseases with high genetic heterogeneity (pathogenic variants identified in over 280 causative genes). The genetic diagnostic rate for IRDs is around 60%, mainly thanks to the routine application of next-generation sequencing (NGS) approaches such as extensive gene panels or whole exome analyses. Whole-genome sequencing (WGS) has been reported to improve this diagnostic rate by revealing elusive variants, such as structural variants (SVs) and deep intronic variants (DIVs). We performed WGS on 33 unsolved cases with suspected autosomal recessive IRD, aiming to identify causative genetic variants in non-coding regions or to detect SVs that were unexplored in the initial screening. Most of the selected cases (30 of 33, 90.9%) carried monoallelic pathogenic variants in genes associated with their clinical presentation, hence we first analyzed the non-coding regions of these candidate genes. Whenever additional pathogenic variants were not identified with this approach, we extended the search for SVs and DIVs to all IRD-associated genes. Overall, we identified the missing causative variants in 11 patients (11 of 33, 33.3%). These included three DIVs in ABCA4, CEP290 and RPGRIP1; one non-canonical splice site (NCSS) variant in PROM1 and three SVs (large deletions) in EYS, PCDH15 and USH2A. For the previously unreported DIV in CEP290 and for the NCCS variant in PROM1, we confirmed the effect on splicing by reverse transcription (RT)-PCR on patient-derived RNA. This study demonstrates the power and clinical utility of WGS as an all-in-one test to identify disease-causing variants missed by standard NGS diagnostic methodologies.
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Doenças Retinianas , Sequenciamento Completo do Genoma , Humanos , Doenças Retinianas/genética , Doenças Retinianas/diagnóstico , Masculino , Feminino , Itália , Criança , Adulto , Adolescente , Predisposição Genética para Doença/genética , Proteínas do Citoesqueleto/genética , Pré-Escolar , Caderinas/genética , Mutação , Variação Genética/genética , Sequenciamento de Nucleotídeos em Larga Escala , Proteínas Relacionadas a Caderinas , Adulto Jovem , Transportadores de Cassetes de Ligação de ATP/genética , Pessoa de Meia-Idade , Proteínas do Olho/genética , Antígenos de Neoplasias/genética , Linhagem , Proteínas de Ciclo CelularRESUMO
Inherited macular dystrophies (iMDs) are a group of genetic disorders, which affect the central region of the retina. To investigate the genetic basis of iMDs, we used single-molecule Molecular Inversion Probes to sequence 105 maculopathy-associated genes in 1352 patients diagnosed with iMDs. Within this cohort, 39.8% of patients were considered genetically explained by 460 different variants in 49 distinct genes of which 73 were novel variants, with some affecting splicing. The top five most frequent causative genes were ABCA4 (37.2%), PRPH2 (6.7%), CDHR1 (6.1%), PROM1 (4.3%) and RP1L1 (3.1%). Interestingly, variants with incomplete penetrance were revealed in almost one-third of patients considered solved (28.1%), and therefore, a proportion of patients may not be explained solely by the variants reported. This includes eight previously reported variants with incomplete penetrance in addition to CDHR1:c.783G>A and CNGB3:c.1208G>A. Notably, segregation analysis was not routinely performed for variant phasing-a limitation, which may also impact the overall diagnostic yield. The relatively high proportion of probands without any putative causal variant (60.2%) highlights the need to explore variants with incomplete penetrance, the potential modifiers of disease and the genetic overlap between iMDs and age-related macular degeneration. Our results provide valuable insights into the genetic landscape of iMDs and warrant future exploration to determine the involvement of other maculopathy genes.
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Degeneração Macular , Humanos , Mutação , Penetrância , Linhagem , Degeneração Macular/genética , Retina , Fenótipo , Transportadores de Cassetes de Ligação de ATP/genética , Proteínas do Olho , Proteínas Relacionadas a Caderinas , Proteínas do Tecido Nervoso/genéticaRESUMO
PURPOSE: To assess the efficacy of voretigene neparvovec (VN) treatment by objective fixation stability and chromatic pupillometry testing in clinical practice. DESIGN: Retrospective cohort study with longitudinal follow-up. SUBJECTS: Twelve patients (aged 7-34 years) with RPE65-related inherited retinal dystrophies were treated at the same center with VN in both eyes. METHODS: Patients treated at the same center with VN were evaluated over a 12-month posttreatment follow-up by subjective and objective tests. Furthermore, patients treated with VN who developed atrophy were compared with those who did not. MAIN OUTCOME MEASURES: Best-corrected visual acuity (BCVA), full-field stimulus threshold test (FST), semiautomated kinetic visual field (SKVF), microperimetry, and chromatic pupillometry over a 12-month follow-up. RESULTS: Significant improvements of BCVA (P < 0.001), SKVF (P < 0.05), and FST (P < 0.001) were already observed 45 days after treatment and were maintained at the 12-month timepoint. Fixation stability, assessed by microperimetry, improved significantly (P < 0.05) after treatment. Chromatic pupillometry showed significant improvements (P < 0.05) at the 6- and 12-month timepoints. The increase in maximum pupillary constriction significantly (P < 0.001) correlated with higher retinal sensitivity in FST. Four patients developed multifocal retinal atrophy in both eyes, detected at the 6-month timepoint, but this atrophy was not generally associated with worse visual function outcomes. CONCLUSIONS: This study explores objective outcomes in order to demonstrate the efficacy of VN treatment in addition to the tests normally performed in clinical practice. Our findings show a significant improvement of retinal function both in subjective assessments, such as BCVA, SKVF, and FST, and in objective measurements of fixation stability and maximum pupillary constriction. Moreover, the significant correlation between maximum pupillary constriction and light sensitivity thresholds corroborates the introduction of chromatic pupillometry as an objective test to better assess treatment outcomes in patients with inherited retinal dystrophies. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found after the references in the Footnotes and Disclosures at the end of this article.
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Distrofias Retinianas , Acuidade Visual , Campos Visuais , cis-trans-Isomerases , Humanos , Masculino , Feminino , Adolescente , Estudos Retrospectivos , Criança , Adulto , Acuidade Visual/fisiologia , Adulto Jovem , Seguimentos , Campos Visuais/fisiologia , cis-trans-Isomerases/genética , Distrofias Retinianas/diagnóstico , Distrofias Retinianas/fisiopatologia , Distrofias Retinianas/genética , Distrofias Retinianas/terapia , Resultado do Tratamento , Terapia Genética/métodos , Testes de Campo VisualRESUMO
Mucopolysaccharidosis type IIIA (MPS IIIA or Sanfilippo syndrome type A) is an autosomal recessive lysosomal storage disorder caused by pathogenic variants in the SGSH gene encoding N-sulfoglucosamine sulfohydrolase, an enzyme involved in the degradation of heparan sulfate. MPS IIIA is typically characterized by neurocognitive decline and hepatosplenomegaly with childhood onset. Here, we report on a 53-year-old male subject initially diagnosed with Usher syndrome for the concurrence of retinitis pigmentosa and sensorineural hearing loss. Clinical exome sequencing identified biallelic missense variants in SGSH, and biochemical assays showed complete deficiency of sulfamidase activity and increased urinary glycosaminoglycan excretion. Reverse phenotyping revealed left ventricle pseudo-hypertrophy, hepatosplenomegaly, bilateral deep white matter hyperintensities upon brain MRI, and decreased cortical metabolic activity by PET-CT. On neuropsychological testing, the proband presented only partial and isolated verbal memory deficits. This case illustrates the power of unbiased, comprehensive genetic testing for the diagnosis of challenging mild or atypical forms of MPS IIIA.
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Mucopolissacaridose III , Síndromes de Usher , Masculino , Humanos , Criança , Pessoa de Meia-Idade , Mucopolissacaridose III/diagnóstico , Mucopolissacaridose III/genética , Hidrolases/genética , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Síndromes de Usher/diagnóstico , Síndromes de Usher/genética , Testes Genéticos , Hepatomegalia/genéticaRESUMO
The ABCA4 gene is the most frequently mutated Mendelian retinopathy-associated gene. Biallelic variants lead to a variety of phenotypes, however, for thousands of cases the underlying variants remain unknown. Here, we aim to shed further light on the missing heritability of ABCA4-associated retinopathy by analyzing a large cohort of macular dystrophy probands. A total of 858 probands were collected from 26 centers, of whom 722 carried no or one pathogenic ABCA4 variant, while 136 cases carried two ABCA4 alleles, one of which was a frequent mild variant, suggesting that deep-intronic variants (DIVs) or other cis-modifiers might have been missed. After single molecule molecular inversion probes (smMIPs)-based sequencing of the complete 128-kb ABCA4 locus, the effect of putative splice variants was assessed in vitro by midigene splice assays in HEK293T cells. The breakpoints of copy number variants (CNVs) were determined by junction PCR and Sanger sequencing. ABCA4 sequence analysis solved 207 of 520 (39.8%) naive or unsolved cases and 70 of 202 (34.7%) monoallelic cases, while additional causal variants were identified in 54 of 136 (39.7%) probands carrying two variants. Seven novel DIVs and six novel non-canonical splice site variants were detected in a total of 35 alleles and characterized, including the c.6283-321C>G variant leading to a complex splicing defect. Additionally, four novel CNVs were identified and characterized in five alleles. These results confirm that smMIPs-based sequencing of the complete ABCA4 gene provides a cost-effective method to genetically solve retinopathy cases and that several rare structural and splice altering defects remain undiscovered in Stargardt disease cases.
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Degeneração Macular , Distrofias Retinianas , Humanos , Células HEK293 , Mutação/genética , Degeneração Macular/genética , Distrofias Retinianas/genética , Análise de Sequência , Transportadores de Cassetes de Ligação de ATP/genéticaRESUMO
BACKGROUND: Non-syndromic inherited retinal dystrophies (IRDs) such as retinitis pigmentosa or Leber congenital amaurosis generally manifest between early childhood and late adolescence, imposing profound long-term impacts as a result of vision impairment or blindness. IRDs are highly heterogeneous, with often overlapping symptoms among different IRDs, and achieving a definite diagnosis is challenging. This narrative review provides a clinical overview of the non-syndromic generalized photoreceptor dystrophies, particularly retinitis pigmentosa and Leber congenital amaurosis. The clinical investigations and genetic testing needed to establish a diagnosis are outlined, and current management approaches are discussed, focusing on the importance of the involvement of an interdisciplinary team from diagnosis and initial care to long-term follow-up and support. RESULTS: The effective management of IRDs requires a multidisciplinary, and ideally interdisciplinary, team of experts knowledgeable about IRDs, with experienced professionals from fields as diverse as ophthalmology, neuropsychiatry, psychology, neurology, genetics, orthoptics, developmental therapy, typhlology, occupational therapy, otolaryngology, and orientation and mobility specialties. Accurate clinical diagnosis encompasses a range of objective and subjective assessments as a prerequisite for the genetic testing essential in establishing an accurate diagnosis necessary for the effective management of IRDs, particularly in the era of gene therapies. Improvements in genome sequencing techniques, such as next-generation sequencing, have greatly facilitated the complex process of determining IRD-causing gene variants and establishing a molecular diagnosis. Genetic counseling is essential to help the individual and their family understand the condition, the potential risk for offspring, and the implications of a diagnosis on visual prognosis and treatment options. Psychological support for patients and caregivers is important at all stages of diagnosis, care, and rehabilitation and is an essential part of the multidisciplinary approach to managing IRDs. Effective communication throughout is essential, and the patient and caregivers' needs and expectations must be acknowledged and discussed. CONCLUSION: As IRDs can present at an early age, clinicians need to be aware of the clinical signs suggesting visual impairment and follow up with multidisciplinary support for timely diagnoses to facilitate appropriate therapeutic or rehabilitation intervention to minimize vision loss.
Assuntos
Amaurose Congênita de Leber , Distrofias Retinianas , Retinose Pigmentar , Adolescente , Humanos , Pré-Escolar , Amaurose Congênita de Leber/diagnóstico , Amaurose Congênita de Leber/genética , Amaurose Congênita de Leber/terapia , Distrofias Retinianas/diagnóstico , Distrofias Retinianas/genética , Distrofias Retinianas/terapia , Retinose Pigmentar/diagnóstico , Retinose Pigmentar/genética , Retinose Pigmentar/terapia , Testes Genéticos , Terapia Genética , MutaçãoRESUMO
OBJECTIVES: VPS35 and VPS13 have been associated with Parkinson's disease (PD), and their shared phenotype in yeast when reduced in function is abnormal vacuolar transport. We aim to test if additional potentially deleterious variants in other genes that share this phenotype can modify the risk for PD. METHODS: 77 VPS and VPS-related genes were analyzed using whole-genome-sequencing data from 202 PD patients of Ashkenazi Jewish (AJ) ancestry. Filtering was done based on quality and functionality scores. Ten variants in nine genes were further genotyped in 1200 consecutively recruited unrelated AJ-PD patients, and allele frequencies and odds ratio calculated compared to gnomAD-AJ-non-neuro database, in un-stratified (n = 1200) and stratified manner (LRRK2-G2019S-PD patients (n = 145), GBA-PD patients (n = 235), and non-carriers of these mutations (NC, n = 787)). RESULTS: Five variants in PIK3C3, VPS11, AP1G2, HGS and VPS13D were significantly associated with PD-risk. PIK3C3-R768W showed a significant association in an un-stratified (all PDs) analysis, as well as in stratified (LRRK2, GBA, and NC) analyses (Odds ratios = 2.71, 5.32, 3.26. and 2.19 with p = 0.0015, 0.002, 0.0287, and 0.0447, respectively). AP1G2-R563W was significantly associated in LRRK2-carriers (OR = 3.69, p = 0.006) while VPS13D-D2932N was significantly associated in GBA-carriers (OR = 5.45, p = 0.0027). VPS11-C846G and HGS-S243Y were significantly associated in NC (OR = 2.48 and 2.06, with p = 0.022 and 0.0163, respectively). CONCLUSIONS: Variants in genes involved in vesicle-mediated protein transport and recycling pathways, including autophagy and mitophagy, may differentially modify PD-risk in LRRK2-carriers, GBA carriers, or NC. Specifically, PIK3C3-R768W is a PD-risk allele, with the highest effect size in LRRK2-G2019S carriers. These results suggest oligogenic effect that may depends on the genetic background of the patient. An unbiased burden of mutations approach in these genes should be evaluated in additional PD and control groups. The mechanisms by which these novel variants interact and increase PD-risk should be researched in depth for better tailoring therapeutic intervention for PD prevention or slowing disease progression.
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Doença de Parkinson , Humanos , Doença de Parkinson/genética , Genótipo , Heterozigoto , Mutação , Fenótipo , Glucosilceramidase/genética , Proteínas/genéticaRESUMO
BACKGROUND: Inherited retinal diseases (IRD) are genetically heterogeneous disorders that cause the dysfunction or loss of photoreceptor cells and ultimately lead to blindness. To date, next-generation sequencing procedures fail to detect pathogenic sequence variants in coding regions of known IRD disease genes in about 30-40% of patients. One of the possible explanations for this missing heritability is the presence of yet unidentified transcripts of known IRD genes. Here, we aimed to define the transcript composition of IRD genes in the human retina by a meta-analysis of publicly available RNA-seq datasets using an ad-hoc designed pipeline. RESULTS: We analysed 218 IRD genes and identified 5,054 transcripts, 3,367 of which were not previously reported. We assessed their putative expression levels and focused our attention on 435 transcripts predicted to account for at least 5% of the expression of the corresponding gene. We looked at the possible impact of the newly identified transcripts at the protein level and experimentally validated a subset of them. CONCLUSIONS: This study provides an unprecedented, detailed overview of the complexity of the human retinal transcriptome that can be instrumental in contributing to the resolution of some cases of missing heritability in IRD patients.
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Doenças Retinianas , Transcriptoma , Humanos , Retina/metabolismo , Doenças Retinianas/genética , Doenças Retinianas/diagnóstico , Doenças Retinianas/metabolismo , MutaçãoRESUMO
Introduction: Retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA) are two groups of inherited retinal diseases (IRDs) where the rod photoreceptors degenerate followed by the cone photoreceptors of the retina. A genetic diagnosis for IRDs is challenging since >280 genes are associated with these conditions. While whole exome sequencing (WES) is commonly used by diagnostic facilities, the costs and required infrastructure prevent its global applicability. Previous studies have shown the cost-effectiveness of sequence analysis using single molecule Molecular Inversion Probes (smMIPs) in a cohort of patients diagnosed with Stargardt disease and other maculopathies. Methods: Here, we introduce a smMIPs panel that targets the exons and splice sites of all currently known genes associated with RP and LCA, the entire RPE65 gene, known causative deep-intronic variants leading to pseudo-exons, and part of the RP17 region associated with autosomal dominant RP, by using a total of 16,812 smMIPs. The RP-LCA smMIPs panel was used to screen 1,192 probands from an international cohort of predominantly RP and LCA cases. Results and discussion: After genetic analysis, a diagnostic yield of 56% was obtained which is on par with results from WES analysis. The effectiveness and the reduced costs compared to WES renders the RP-LCA smMIPs panel a competitive approach to provide IRD patients with a genetic diagnosis, especially in countries with restricted access to genetic testing.
RESUMO
Here we describe three patients with neurodevelopmental disorders characterized by mild-to-moderate intellectual disability, mildly dysmorphic features, and hirsutism, all of which carry de novo sequence variants in the WW domain-containing adaptor of the coiled-coil (WAC) gene; two of these-c.167delA, p.(Asn56I1efs*136) and c.1746G>C, p.(Gln582His)-are novel pathogenic variants, and the third-c.1837C>T, p(Arg613*)-has been previously described. Diseases associated with WAC include DeSanto-Shinawi syndrome; to date, de novo heterozygous constitutional pathogenic WAC variants have caused a syndromic form of intellectual disability and mild dysmorphic features in 33 patients, yet potential associations with other clinical manifestations, such as oligomenorrhea and hyperandrogenism, remain unknown, because the phenotypic spectrum of the condition has not yet been delineated. The patient bearing the novel c.167delA WAC gene variant presented a normal psychomotor development, oligomenorrhea, hyperandrogenism, and hirsutism, and hirsutism was also observed in the patient with the c.1746G>C WAC gene variant. Hypertrichosis and hirsutism have been described in nine DeSanto-Shinawi patients, only in 17 of the 33 aforementioned patients thus far reported this aspect, and no hormonal-pattern data are available. In conclusion, we note that the pathogenic c.167delA WAC variant may be associated with a mild phenotype; and in addition to the neurodevelopmental problems nearly all DeSanto-Shinawi patients experience (i.e., intellectual disability and/or developmental delay), we recommend the addition of mild dysmorphic features, hirsutism, and hypertrichosis to this clinical presentation.
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Hiperandrogenismo , Hipertricose , Deficiência Intelectual , Humanos , Feminino , Deficiência Intelectual/genética , Hirsutismo/genética , Hipertricose/genética , Oligomenorreia , FenótipoRESUMO
Inherited retinal diseases (IRDs) are the leading cause of vision loss in the working-age population. We performed a retrospective epidemiological study to determine the genetic basis of IRDs in a large Italian cohort (n = 2790) followed at a single referral center. We provided, mainly by next generation sequencing, potentially conclusive molecular diagnosis for 2036 patients (from 1683 unrelated families). We identified a total of 1319 causative sequence variations in 132 genes, including 353 novel variants, and 866 possibly actionable genotypes for therapeutic approaches. ABCA4 was the most frequently mutated gene (n = 535; 26.3% of solved cases), followed by USH2A (n = 228; 11.2%) and RPGR (n = 102; 5.01%). The other 129 genes had a lower contribution to IRD pathogenesis (e.g. CHM 3.5%, RHO 3.5%; MYO7A 3.4%; CRB1 2.7%; RPE65 2%, RP1 1.8%; GUCY2D 1.7%). Seventy-eight genes were mutated in five patients or less. Mitochondrial DNA variants were responsible for 2.1% of cases. Our analysis confirms the complex genetic etiology of IRDs and reveals the high prevalence of ABCA4 and USH2A mutations. This study also uncovers genetic associations with a spectrum of clinical subgroups and highlights a valuable number of cases potentially eligible for clinical trials and, ultimately, for molecular therapies.
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Doenças Retinianas , Humanos , Epidemiologia Molecular , Estudos Retrospectivos , Doenças Retinianas/epidemiologia , Doenças Retinianas/genética , Retina , Itália/epidemiologia , Proteínas do Olho/genética , Transportadores de Cassetes de Ligação de ATP/genética , Proteínas de Membrana/genética , Proteínas do Tecido NervosoRESUMO
To report quantitative retinal changes assessed by spectral-domain optical coherence tomography (SD-OCT) in children treated with voretigene neparvovec (VN) at a single center in Italy. Retrospective review of six consecutive pediatric patients with biallelic RPE65-related dystrophy treated bilaterally with VN. SD-OCT scans were analyzed to extract Early Treatment Diabetic Retinopathy Study (ETDRS) thickness maps of the whole retina and the outer nuclear layer (ONL). Changes in visual function were assessed by best-corrected visual acuity (BCVA) and retinal morphology at Days 30/45 and 180. BCVA significantly improved at Day 30/45 and 6 months (both P < 0.001). Central foveal retinal thickness and central foveal ONL thickness tended to increase (6.4 ± 19.2 µm; P = 0.080 and 3.42 ± 7.68 µm; P = 0.091, respectively). ONL thickness of the internal ETDRS-ring significantly increased at day 30/45 (4.7 ± 8.4 µm; P < 0.001) and day 180 (5.0 ± 5.7 µm; P = 0.009). Intra-operative foveal detachment was not associated with a higher function gain in terms of BCVA, but with a mild thinning of foveal ONL after treatment. The improvement of BCVA and thickening of the ONL layer suggest that improvement of visual acuity could be related to partial recovery of retinal morphology in the perifoveal ring.
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Retina , Distrofias Retinianas , Humanos , Criança , Retina/diagnóstico por imagem , Fóvea Central/diagnóstico por imagem , Tomografia de Coerência Óptica/métodos , Acuidade Visual , Distrofias Retinianas/genética , Distrofias Retinianas/terapia , Estudos RetrospectivosRESUMO
Inherited retinal diseases (IRDs) are a group of diseases whose common landmark is progressive photoreceptor loss. The development of gene-specific therapies for IRDs is hampered by their wide genetic heterogeneity. Mitochondrial dysfunction is proving to constitute one of the key pathogenic events in IRDs; hence, approaches that enhance mitochondrial activities have a promising therapeutic potential for these conditions. We previously reported that miR-181a/b downregulation boosts mitochondrial turnover in models of primary retinal mitochondrial diseases. Here, we show that miR-181a/b silencing has a beneficial effect also in IRDs. In particular, the injection in the subretinal space of an adeno-associated viral vector (AAV) that harbors a miR-181a/b inhibitor (sponge) sequence (AAV2/8-GFP-Sponge-miR-181a/b) improves retinal morphology and visual function both in models of autosomal dominant (RHO-P347S) and of autosomal recessive (rd10) retinitis pigmentosa. Moreover, we demonstrate that miR-181a/b downregulation modulates the level of the mitochondrial fission-related protein Drp1 and rescues the mitochondrial fragmentation in RHO-P347S photoreceptors. Overall, these data support the potential use of miR-181a/b downregulation as an innovative mutation-independent therapeutic strategy for IRDs, which can be effective both to delay disease progression and to aid gene-specific therapeutic approaches.
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MicroRNAs , Retinose Pigmentar , Humanos , Regulação para Baixo , Retina/patologia , Retinose Pigmentar/genética , Retinose Pigmentar/terapia , Retinose Pigmentar/metabolismo , Mutação , MicroRNAs/genética , MicroRNAs/metabolismoRESUMO
Retinal photoreceptors have a distinct transcriptomic profile compared to other neuronal subtypes, likely reflecting their unique cellular morphology and function in the detection of light stimuli by way of the ciliary outer segment. We discovered a layer of this molecular specialization by revealing that the vertebrate retina expresses the largest number of tissue-enriched microexons of all tissue types. A subset of these microexons is included exclusively in photoreceptor transcripts, particularly in genes involved in cilia biogenesis and vesicle-mediated transport. This microexon program is regulated by Srrm3, a paralog of the neural microexon regulator Srrm4. Despite the fact that both proteins positively regulate retina microexons in vitro, only Srrm3 is highly expressed in mature photoreceptors. Its deletion in zebrafish results in widespread down-regulation of microexon inclusion from early developmental stages, followed by other transcriptomic alterations, severe photoreceptor defects, and blindness. These results shed light on the transcriptomic specialization and functionality of photoreceptors, uncovering unique cell type-specific roles for Srrm3 and microexons with implications for retinal diseases.
Assuntos
Proteínas , Segmento Externo das Células Fotorreceptoras da Retina , Fatores de Processamento de Serina-Arginina , Visão Ocular , Animais , Éxons , Deleção de Genes , Humanos , Proteínas/genética , Proteínas/fisiologia , Segmento Externo das Células Fotorreceptoras da Retina/metabolismo , Fatores de Processamento de Serina-Arginina/genética , Fatores de Processamento de Serina-Arginina/fisiologia , Transcriptoma , Visão Ocular/genética , Visão Ocular/fisiologia , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento , Proteínas de Peixe-Zebra/genéticaRESUMO
Purpose: To investigate the course of inherited retinal degenerations (IRD) due to mutations in the RPE65 gene. Methods: This longitudinal multicentric retrospective chart-review study was designed to collect best corrected visual acuity (BCVA), Goldman visual field, optical coherence tomography (OCT), and electroretinography (ERG) measurements. The data, including imaging, were collected using an electronic clinical research form and were reviewed at a single center to improve consistency. Results: From an overall cohort of 60 Italian patients with RPE65-associated IRD, 43 patients (mean age, 27.8 ± 19.7 years) were included and showed a mean BCVA of 2.0 ± 1.0 logMAR. Time-to-event analysis revealed a median age of 33.8 years and 41.4 years to reach low vision and blindness based on BCVA, respectively. ERG (available for 34 patients) showed undetectable responses in most patients (26; 76.5%). OCT (available for 31 patients) revealed epiretinal membranes in five patients (16.1%). Central foveal thickness significantly decreased with age at a mean annual rate of -0.6%/y (P = 0.044). We identified 43 different variants in the RPE65 gene in the entire cohort. Nine variants were novel. Finally, to assess genotype-phenotype correlations, patients were stratified according to the number of RPE65 loss-of-function (LoF) alleles. Patients without LoF variants showed significantly (P < 0.05) better BCVA compared to patients with one or two LoF alleles. Conclusions: We described the natural course of RPE65-associated IRD in an Italian cohort showing for the first time a specific genotype-phenotype association. Our findings can contribute to a better management of RPE65-associated IRD patients.
Assuntos
DNA/genética , Mutação , Distrofias Retinianas/genética , Acuidade Visual , Campos Visuais , cis-trans-Isomerases/genética , Adolescente , Adulto , Criança , Pré-Escolar , Análise Mutacional de DNA , Eletrorretinografia , Feminino , Estudos de Associação Genética , Genótipo , Humanos , Incidência , Itália/epidemiologia , Masculino , Pessoa de Meia-Idade , Fenótipo , Distrofias Retinianas/diagnóstico , Distrofias Retinianas/epidemiologia , Estudos Retrospectivos , Tomografia de Coerência Óptica , Adulto Jovem , cis-trans-Isomerases/metabolismoRESUMO
Homozygous deletions (HDs) may be the cause of rare diseases and cancer, and their discovery in targeted sequencing is a challenging task. Different tools have been developed to disentangle HD discovery but a sensitive caller is still lacking. We present VarGenius-HZD, a sensitive and scalable algorithm that leverages breadth-of-coverage for the detection of rare homozygous and hemizygous single-exon deletions (HDs). To assess its effectiveness, we detected both real and synthetic rare HDs in fifty exomes from the 1000 Genomes Project obtaining higher sensitivity in comparison with state-of-the-art algorithms that each missed at least one event. We then applied our tool on targeted sequencing data from patients with Inherited Retinal Dystrophies and solved five cases that still lacked a genetic diagnosis. We provide VarGenius-HZD either stand-alone or integrated within our recently developed software, enabling the automated selection of samples using the internal database. Hence, it could be extremely useful for both diagnostic and research purposes.
Assuntos
Variações do Número de Cópias de DNA/genética , Análise de Sequência de DNA/métodos , Deleção de Sequência/genética , Algoritmos , Animais , Sequência de Bases/genética , Exoma/genética , Éxons/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , HumanosRESUMO
Purpose: To present visual outcomes of the first two Italian patients with RPE65-related inherited retinal dystrophy (RPE65-IRD) treated with voretigene neparvovec (VN). Methods: Two pediatric patients with RPE65-IRD were treated with VN in both eyes. Patients were evaluated by best-corrected visual acuity (BCVA), full-field stimulus threshold (FST) test, semiautomated kinetic visual field (SKVF), microperimetry, and chromatic pupillometry over 6 months. Results: No complications occurred in the first patient, whereas in the second a subretinal hemorrhage was observed in the first treated eye, and excessive resistance to drug injection occurred during treatment of the second eye. BCVA improved by at least one Early Treatment Diabetic Retinopathy Study line in all treated eyes. The FST test and SKVF showed clinically significant improvements in all eyes (i.e., change of light sensitivity > 10 decibels; area enlargement of at least 20%). Moreover, microperimetry showed better fixation stability. Finally, chromatic pupillometry showed increases in pupillary constriction that ranged from 10% to 20%. All visual changes remained stable during follow-up. Conclusions: The first VN treatments in two pediatric Italian patients in clinical practice showed significant improvements in visual outcomes, even in the case of surgical complications, which spontaneously recovered without sequelae. Translational Relevance: These findings with VN in patients with RPE65-IRD confirm the results of clinical trials.
Assuntos
Terapia Genética , Distrofias Retinianas , Criança , Olho , Humanos , Itália , Distrofias Retinianas/terapia , Campos VisuaisRESUMO
Pathogenic variants in the MKS1 gene are responsible for a ciliopathy with a wide spectrum of clinical manifestations ranging from Meckel and Joubert syndrome (JBTS) to Bardet-Biedl syndrome, and involving the central nervous system, liver, kidney, skeleton, and retina. We report a 39-year-old male individual presenting with isolated Retinitis Pigmentosa (RP), as assessed by full ophthalmological evaluation including Best-Corrected Visual Acuity measurements, fundus examination, Goldmann Visual Field test, and full-field Electroretinography. A clinical exome identified biallelic nonsense variants in MKS1 that prompted post-genotyping investigations for systemic abnormalities of ciliopathy. Brain magnetic resonance imaging revealed malformations of the posterior cranial fossa with the 'molar tooth sign' and cerebellar folia dysplasia, which are both distinctive features of JBTS. No other organ or skeletal abnormalities were detected. This case illustrates the power of clinical exome for the identification of the mildest forms of a disease spectrum, such as a mild JBTS with RP in the presented case of an individual carrying biallelic truncating variants in MKS1.