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Advances in DNA sequencing technologies have revolutionised rare disease diagnostics and have led to a dramatic increase in the volume of available genomic data. A key challenge that needs to be overcome to realise the full potential of these technologies is that of precisely predicting the effect of genetic variants on molecular and organismal phenotypes. Notably, despite recent progress, there is still a lack of robust in silico tools that accurately assign clinical significance to variants. Genetic alterations in the CACNA1F gene are the commonest cause of X-linked incomplete Congenital Stationary Night Blindness (iCSNB), a condition associated with non-progressive visual impairment. We combined genetic and homology modelling data to produce CACNA1F-vp, an in silico model that differentiates disease-implicated from benign missense CACNA1F changes. CACNA1F-vp predicts variant effects on the structure of the CACNA1F encoded protein (a calcium channel) using parameters based upon changes in amino acid properties; these include size, charge, hydrophobicity, and position. The model produces an overall score for each variant that can be used to predict its pathogenicity. CACNA1F-vp outperformed four other tools in identifying disease-implicated variants (area under receiver operating characteristic and precision recall curves = 0.84; Matthews correlation coefficient = 0.52) using a tenfold cross-validation technique. We consider this protein-specific model to be a robust stand-alone diagnostic classifier that could be replicated in other proteins and could enable precise and timely diagnosis.

Purpose: A key property to consider in all genetic tests is clinical utility, the ability of the test to influence patient management and health outcomes. Here we assess the current clinical utility of genetic testing in diverse pediatric inherited eye disorders (IEDs)., Methods: Two hundred one unrelated children (0-5 years old) with IEDs were ascertained through the database of the North West Genomic Laboratory Hub, Manchester, UK. The cohort was collected over a 7-year period (2011-2018) and included 74 children with bilateral cataracts, 8 with bilateral ectopia lentis, 28 with bilateral anterior segment dysgenesis, 32 with albinism, and 59 with inherited retinal disorders. All participants underwent panel-based genetic testing., Results: The diagnostic yield of genetic testing for the cohort was 64% (ranging from 39% to 91% depending on the condition). The test result led to altered management (including preventing additional investigations or resulting in the introduction of personalized surveillance measures) in 33% of probands (75% for ectopia lentis, 50% for cataracts, 33% for inherited retinal disorders, 7% for anterior segment dysgenesis, 3% for albinism)., Conclusion: Genetic testing helped identify an etiological diagnosis in the majority of preschool children with IEDs. This prevented additional unnecessary testing and provided the opportunity for anticipatory guidance in significant subsets of patients.

Incremental advances in the field of retinal genetics have transformed our understanding of inherited retinal disorders and have led to the development of powerful diagnostic tests and promising gene-based therapies. Despite this, successful integration of these developments into routine healthcare is frequently ineffective. Providing robust evidence of benefit can accelerate the implementation of clinical genetic interventions. For example, the adoption of a genetic test is much more likely when the test's clinical utility (i.e. its ability to influence management and health outcomes) has been clearly demonstrated. However, accruing such evidence for rare conditions like inherited retinal disorders is challenging. Conducting sufficiently powered studies requires both efficient study designs and large-scale, international collaboration. Reaching all populations and as many affected individuals as possible is key. Equally important are efforts to precisely and consistently capture phenotypic information, including natural history data. This article summarizes some of the current obstacles to implemen-tation and discusses approaches to overcome these barriers., (© 2019 S. Karger AG, Basel.)

Purpose: To assess the clinical usefulness of genetic testing in a pediatric population with inherited retinal disease (IRD)., Design: Single-center retrospective case series., Participants: Eighty-five unrelated children with a diagnosis of isolated or syndromic IRD who were referred for clinical genetic testing between January 2014 and July 2016., Methods: Participants underwent a detailed ophthalmic examination, accompanied by electrodiagnostic testing (EDT) and dysmorphologic assessment where appropriate. Ocular and extraocular features were recorded using Human Phenotype Ontology terms. Subsequently, multigene panel testing (105 or 177 IRD-associated genes) was performed in an accredited diagnostic laboratory, followed by clinical variant interpretation., Main Outcome Measures: Diagnostic yield and clinical usefulness of genetic testing., Results: Overall, 78.8% of patients (n = 67) received a probable molecular diagnosis; 7.5% (n = 5) of these had autosomal dominant disease, 25.4% (n = 17) had X-linked disease, and 67.2% (n = 45) had autosomal recessive disease. In a further 5.9% of patients (n = 5), a single heterozygous ABCA4 variant was identified; all these participants had a spectrum of clinical features consistent with ABCA4 retinopathy. Most participants (84.7%; n = 72) had undergone EDT and 81.9% (n = 59) of these patients received a probable molecular diagnosis. The genes most frequently mutated in the present cohort were CACNA1F and ABCA4, accounting for 14.9% (n = 10) and 11.9% (n = 8) of diagnoses respectively. Notably, in many cases, genetic testing helped to distinguish stationary from progressive IRD subtypes and to establish a precise diagnosis in a timely fashion., Conclusions: Multigene panel testing pointed to a molecular diagnosis in 84.7% of children with IRD. The diagnostic yield in the study population was significantly higher compared with that in previously reported unselected IRD cohorts. Approaches similar to the one described herein are expected to become a standard component of care in pediatric ophthalmology. We propose the introduction of genetic testing early in the diagnostic pathway in children with clinical and/or electrophysiologic findings, suggestive of IRD., (Copyright © 2017 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.)

Although a common cause of disease, copy number variants (CNVs) have not routinely been identified from next-generation sequencing (NGS) data in a clinical context. This study aimed to examine the sensitivity and specificity of a widely used software package, ExomeDepth, to identify CNVs from targeted NGS data sets. We benchmarked the accuracy of CNV detection using ExomeDepth v1.1.6 applied to targeted NGS data sets by comparison to CNV events detected through whole-genome sequencing for 25 individuals and determined the sensitivity and specificity of ExomeDepth applied to these targeted NGS data sets to be 100% and 99.8%, respectively. To define quality assurance metrics for CNV surveillance through ExomeDepth, we undertook simulation of single-exon (n=1000) and multiple-exon heterozygous deletion events (n=1749), determining a sensitivity of 97% (n=2749). We identified that the extent of sequencing coverage, the inter- and intra-sample variability in the depth of sequencing coverage and the composition of analysis regions are all important determinants of successful CNV surveillance through ExomeDepth. We then applied these quality assurance metrics during CNV surveillance for 140 individuals across 12 distinct clinical areas, encompassing over 500 potential rare disease diagnoses. All 140 individuals lacked molecular diagnoses after routine clinical NGS testing, and by application of ExomeDepth, we identified 17 CNVs contributing to the cause of a Mendelian disorder. Our findings support the integration of CNV detection using ExomeDepth v1.1.6 with routine targeted NGS diagnostic services for Mendelian disorders. Implementation of this strategy increases diagnostic yields and enhances clinical care.

Defects in primary cilium biogenesis underlie the ciliopathies, a growing group of genetic disorders. We describe a whole-genome siRNA-based reverse genetics screen for defects in biogenesis and/or maintenance of the primary cilium, obtaining a global resource. We identify 112 candidate ciliogenesis and ciliopathy genes, including 44 components of the ubiquitin-proteasome system, 12 G-protein-coupled receptors, and 3 pre-mRNA processing factors (PRPF6, PRPF8 and PRPF31) mutated in autosomal dominant retinitis pigmentosa. The PRPFs localize to the connecting cilium, and PRPF8- and PRPF31-mutated cells have ciliary defects. Combining the screen with exome sequencing data identified recessive mutations in PIBF1, also known as CEP90, and C21orf2, also known as LRRC76, as causes of the ciliopathies Joubert and Jeune syndromes. Biochemical approaches place C21orf2 within key ciliopathy-associated protein modules, offering an explanation for the skeletal and retinal involvement observed in individuals with C21orf2 variants. Our global, unbiased approaches provide insights into ciliogenesis complexity and identify roles for unanticipated pathways in human genetic disease.

Non-traumatic ectopia lentis can be isolated or herald an underlying multisystemic disorder. Technological advances have revolutionized genetic testing for many ophthalmic disorders, and this study aims to provide insights into the clinical utility of genetic analysis in paediatric ectopia lentis. Children that underwent lens extraction for ectopia lentis between 2013 and 2017 were identified, and gene panel testing findings and surgical outcomes were collected. Overall, 10/11 cases received a probable molecular diagnosis. Genetic variants were identified in four genes: FBN1 (associated with Marfan syndrome and cardiovascular complications; n = 6), ADAMTSL4 (associated with non-syndromic ectopia lentis; n = 2), LTBP2 (n = 1) and ASPH (n = 1). Parents appeared unaffected in 6/11 cases; the initial presentation of all six of these children was to an ophthalmologist, and only 2/6 had FBN1 variants. Notably, 4/11 cases required surgery before the age of 4 years, and only one of these children carried an FBN1 variant. In summary, in this retrospective cohort study, panel-based genetic testing pointed to a molecular diagnosis in >90% of paediatric ectopia lentis cases requiring surgery. In a subset of study participants, genetic analysis revealed changes in genes that have not been linked to extraocular manifestations and highlighted that extensive systemic investigations were not required in these individuals. We propose the introduction of genetic testing early in the diagnostic pathway in children with ectopia lentis. [ABSTRACT FROM AUTHOR]

Purpose: Congenital stationary night blindness (CSNB) is a heterogeneous group of Mendelian retinal disorders that present in childhood. Biallelic variants altering the protein‐coding region of the TRPM1 gene are one of the commonest causes of CSNB. Here, we report the clinical and genetic findings in 10 unrelated individuals with TRPM1‐retinopathy. Methods: Study subjects were recruited through a tertiary clinical ophthalmic genetic service at Manchester, UK. All participants underwent visual electrodiagnostic testing and panel‐based genetic analysis. Results: Study subjects had a median age of 8 years (range: 3–20 years). All probands were myopic and had electroretinographic findings in keeping with complete CSNB. Notably, three probands reported no night vision problems. Fourteen different disease‐associated TRPM1 variants were detected. One individual was homozygous for the NM_001252024.2 (TRPM1):c.965 + 29G>A variant and a mini‐gene assay highlighted that this change results in mis‐splicing and premature protein termination. Additionally, two unrelated probands who had CSNB and mild neurodevelopmental abnormalities were found to carry a 15q13.3 microdeletion. This copy number variant encompasses seven genes, including TRPM1, and was encountered in the heterozygous state and in trans with a missense TRPM1 variant in each case. Conclusion: Our findings highlight the importance of comprehensive genomic analysis, beyond the exons and protein‐coding regions of genes, for individuals with CSNB. When this characteristic retinal phenotype is accompanied by extraocular findings (including learning and/or behavioural difficulties), a 15q13.3 microdeletion should be suspected. Focused analysis (e.g. microarray testing) is recommended to look for large‐scale deletions encompassing TRPM1 in patients with CSNB and neurodevelopmental abnormalities. [ABSTRACT FROM AUTHOR]

Agnathia-otocephaly complex is a malformation characterized by absent/hypoplastic mandible and abnormally positioned ears. Mutations in two genes, PRRX1 and OTX2, have been described in a small number of families with this disorder. We performed clinical and genetic testing in an additional family. The proband is a healthy female with a complicated pregnancy history that includes two offspring diagnosed with agnathia-otocephaly during prenatal ultrasound scans. Exome sequencing was performed in fetal DNA from one of these two offspring revealing a heterozygous duplication in OTX2: c.271_273dupCAG, p.(Gln91dup). This change leads to the insertion of a glutamine within the OTX2 homeodomain region, and is predicted to alter this signaling molecule's ability to interact with DNA. The same variant was also identified in the proband's clinically unaffected 38-year-old husband and their 9-year-old daughter, who presented with a small mandible, normal ears and velopharyngeal insufficiency due to a short hemi-palate. This unusual presentation of OTX2-related disease suggests that OTX2 might have a role in palatal hypoplasia cases. A previously unreported OTX2 variant associated with extreme intrafamilial variability is described and the utility of exome sequencing as a tool to confirm the diagnosis of agnathia-otocephaly and to inform the reproductive decisions of affected families is highlighted. [ABSTRACT FROM AUTHOR]