Pathogenicity Reclassification of RPE65 Missense Variants Related to Leber Congenital Amaurosis and Early-Onset Retinal Dystrophy

A challenge in molecular diagnosis and genetic counseling is the interpretation of variants of uncertain significance. Proper pathogenicity classification of new variants is important for the conclusion of molecular diagnosis and the medical management of patient treatments. The purpose of this study was to reclassify two RPE65 missense variants, c.247T>C (p.Phe83Leu) and c.560G>A (p.Gly187Glu), found in Brazilian families. To achieve this aim, we reviewed the sequencing data of a 224-gene retinopathy panel from 556 patients (513 families) with inherited retinal dystrophies. Five patients with p.Phe83Leu and seven with p.Gly187Glu were selected and their families investigated. To comprehend the pathogenicity of these variants, we evaluated them based on the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) classification guidelines. Initially, these RPE65 variants met only three pathogenic criteria: (i) absence or low frequency in the population, (ii) several missense pathogenic RPE65 variants, and (iii) 15 out of 16 lines of computational evidence supporting them as damaging, which together allowed the variants to be classified as uncertain significance. Two other pieces of evidence were accepted after further analysis of these Brazilian families: (i) p.Phe83Leu and p.Gly187Glu segregate with childhood retinal dystrophy within families, and (ii) their prevalence in Leber congenital amaurosis (LCA)/early-onset retinal dystrophy (EORD) patients can be considered higher than in other inherited retinal dystrophy patients. Therefore, these variants can now be classified as likely pathogenic according to ACMG/AMP classification guidelines.

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Molecular Screening of 43 Brazilian Families Diagnosed with Leber Congenital Amaurosis or Early-Onset Severe Retinal Dystrophy

Genes ◽

10.3390/genes8120355 ◽

2017 ◽

Vol 8 (12) ◽

pp. 355 ◽

Cited By ~ 7

Author(s):

Fernanda Porto ◽

Evan Jones ◽

Justin Branch ◽

Zachry Soens ◽

Igor Maia ◽

...

Keyword(s):

Early Onset ◽

Retinal Dystrophy ◽

Molecular Screening ◽

Leber Congenital Amaurosis

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Innate and Autoimmunity in the Pathogenesis of Inherited Retinal Dystrophy

Cells ◽

10.3390/cells9030630 ◽

2020 ◽

Vol 9 (3) ◽

pp. 630 ◽

Cited By ~ 2

Author(s):

T. J. Hollingsworth ◽

Alecia K. Gross

Keyword(s):

Disease Progression ◽

Age Of Onset ◽

Retinal Dystrophy ◽

Inherited Retinal Dystrophies ◽

Retinal Dystrophies ◽

Major Player ◽

Inherited Retinal Dystrophy ◽

Rate Of Progression ◽

Immunohistochemical Methods ◽

Onset Rate

Inherited retinal dystrophies (RDs) are heterogenous in many aspects including genes involved, age of onset, rate of progression, and treatments. While RDs are caused by a plethora of different mutations, all result in the same outcome of blindness. While treatments, both gene therapy-based and drug-based, have been developed to slow or halt disease progression and prevent further blindness, only a small handful of the forms of RDs have treatments available, which are primarily for recessively inherited forms. Using immunohistochemical methods coupled with electroretinography, optical coherence tomography, and fluorescein angiography, we show that in rhodopsin mutant mice, the involvement of both the innate and the autoimmune systems could be a strong contributing factor in disease progression and pathogenesis. Herein, we show that monocytic phagocytosis and inflammatory cytokine release along with protein citrullination, a major player in forms of autoimmunity, work to enhance the progression of RD associated with a rhodopsin mutation.

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Genetic and clinical findings in a Chinese cohort with Leber congenital amaurosis and early onset severe retinal dystrophy

British Journal of Ophthalmology ◽

10.1136/bjophthalmol-2019-314281 ◽

2019 ◽

Vol 104 (7) ◽

pp. 932-937 ◽

Cited By ~ 3

Author(s):

Ke Xu ◽

Yue Xie ◽

Tengyang Sun ◽

Xiaohui Zhang ◽

Chunjie Chen ◽

...

Keyword(s):

Early Onset ◽

Retinal Dystrophy ◽

Case Series ◽

Clinical Findings ◽

Chinese Patients ◽

Pcr Analysis ◽

Common Mutation ◽

Essential Information ◽

Leber Congenital Amaurosis ◽

Targeted Next Generation Sequencing

BackgroundLeber congenital amaurosis (LCA) and early onset severe retinal dystrophy (EOSRD) are clinically and genetically heterogeneous inherited retinal disorders that cause severe visual impairment in children. The objective of this study was to describe the mutation profile and phenotypic characteristics in Chinese patients with LCA or EOSRD.MethodsRetrospective consecutive case series (2010–2017) study was performed in 148 probands (91 with LCA and 57 with EOSRD). All patients underwent ophthalmic evaluation. Mutations were revealed using targeted next-generation sequencing, followed by Sanger DNA-sequencing and real-time quantitative PCR analysis.ResultsWe identified two diseasing-causing mutations in 88 unrelated patients, heterozygous autosomal dominant mutations in 11 probands and X-linked hemizygous mutations in 11 patients, for an overall mutation detection rate of 74.3% (110/148). We detected 158 different disease-causing mutations involving 14 LCA genes, 16 retinitis pigmentosa or cone-rod dystrophy genes and 3 syndromic retinal dystrophy genes. Of these 158 mutations, 98 were novel. The most common mutation was p.Q141X of AIPL1, with a gene-specific allele frequency of 60%. The first five most frequently mutated genes were AIPL1 (11.0%), RPGRIP1 (8.8%) and CEP290, GUCY2D and RPE65 (each 7.7%) in the patients with LCA and RPGR (12.3%), CRB1 (10.5%), RPE65 (10.5%), RDH12 (7.0%) and RP2 (5.3%) in the patients with EOSRD.ConclusionsOur results revealed that the mutation spectrum of patients with LCA differs from that of the patients with EOSRD and established the configuration of the mutation frequencies for each LCA gene in Chinese patients, thereby providing essential information for future genetic counselling and gene therapy.

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An Assessment of the Apex Microarray Technology in Genotyping Patients with Leber Congenital Amaurosis and Early-Onset Severe Retinal Dystrophy

Investigative Opthalmology & Visual Science ◽

10.1167/iovs.07-0207 ◽

2007 ◽

Vol 48 (12) ◽

pp. 5684 ◽

Cited By ~ 35

Author(s):

Robert H. Henderson ◽

Naushin Waseem ◽

Rowan Searle ◽

Jacqueline van der Spuy ◽

Isabelle Russell-Eggitt ◽

...

Keyword(s):

Early Onset ◽

Retinal Dystrophy ◽

Microarray Technology ◽

Leber Congenital Amaurosis

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Leber congenital amaurosis/early-onset severe retinal dystrophy: clinical features, molecular genetics and therapeutic interventions

British Journal of Ophthalmology ◽

10.1136/bjophthalmol-2016-309975 ◽

2017 ◽

Vol 101 (9) ◽

pp. 1147-1154 ◽

Cited By ~ 81

Author(s):

Neruban Kumaran ◽

Anthony T Moore ◽

Richard G Weleber ◽

Michel Michaelides

Keyword(s):

Molecular Genetics ◽

Clinical Features ◽

Early Onset ◽

Retinal Dystrophy ◽

Therapeutic Interventions ◽

Leber Congenital Amaurosis

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SUN-710 Custom Panel to Diagnosis Genetic Endocrine Disorders in a Tertiary Academic Hospital

Journal of the Endocrine Society ◽

10.1210/jendso/bvaa046.1802 ◽

2020 ◽

Vol 4 (Supplement_1) ◽

Author(s):

Lais Cavalca Cardoso ◽

Amanda de Moraes Narcizo ◽

Anna Flavia Figueredo Benedetti ◽

Alexander Augusto Lima Jorge ◽

Barbara Leitao Braga ◽

...

Keyword(s):

Candidate Genes ◽

Molecular Diagnosis ◽

Variant Calling ◽

Endocrine Disorders ◽

Sequencing Data ◽

Academic Hospital ◽

New Findings ◽

Uncertain Significance ◽

Big Picture ◽

Custom Panel

Abstract Next-generation sequencing (NGS) has been transforming the endocrine diagnostic methodology allowing the genetic testing to assume an exploratory role rather than only a confirmatory one. This is possible due to lower costs and increased yield of information. A way to further increase efficiency and sensitivity for variant detection is the use of a sequencing custom panel selecting specific genes for screening. In endocrine disorders, the complex and intricate genotype-phenotype relations and occurrence of diverse comorbidities made the diagnosis challenging. Our aim is to analyze the efficiency of a multigenic panel for molecular diagnosis of endocrine disorders in patients assisted in a tertiary academic hospital, as well as to train academic and medical faculties in the use of molecular tools. Genomic DNA from 282 patients was extracted from blood sample using standard procedures. Sanger method was previously used to screen some candidate genes in half of the patients. The custom panel was designed with 651 genes using the SureDesign tool (Agilent technologies), either associated with the phenotype (OMIM) or candidate genes that englobes developmental (DD), metabolic (MD), and adrenal (AD) disorders. Libraries were prepared with SureSelectXT Target Enrichment kit (Agilent Technologies). The enriched DNA libraries were sequenced in NextSeq 500 (Illumina) with High Output V2 kit (2 x 150 bp). The raw data was aligned to hg19 with BWA-MEM, variant calling was performed using FreeBayes and annotated with ANNOVAR. Filtering took into consideration the rarity (≤1%) of variants in population databases and those in exonic or splice site regions. Variants found were then classified according ACMG/AMP criteria. The categories of Pathogenic (P) and Likely Pathogenic (LP) were considered for molecular diagnosis, while variants of uncertain significance (VUS) were only reported. The average result of 3 runs was: 159Kmm2 of cluster density, 76.5 % of Q30 and 76.6 Gb of data were generated. The mean coverage depth of the targeted regions in panel sequencing data was 237x (SD±110x), with at least 96.3% of the sequenced bases being covered more than 20-fold. Out of the 282 patients, we identified 65 LP/P variants (23%), 22 VUS (8%) and 195 remained undiagnosed (69%). Considering the solved cases, 54 (19.1%) have DD, 6 (2.1%) have MD and 5 (1.8%) have AD. Taking into account that half of the patients had already been previously screened, the data enable new findings in known genes. The application of a multigenic panel aids the training of medical faculty in an academic hospital by showing the big picture of the molecular pathways behind each disorder. This may be particularly helpful considering the higher diagnosis of DD cases. A precise genetic etiology provides improvement in understanding the disease, guides decisions about prevention or treatment, and brings comfort to the affected families.

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