Quality assurance for next-generation sequencing diagnostics of rare neurological diseases in the European Reference Network.

In the past decade, next-generation sequencing (NGS) has revolutionised genetic diagnostics for rare neurological disorders (RND). However, the lack of standardised technical, interpretative, and reporting standards poses a challenge for ensuring consistent and high-quality diagnostics globally. To address this, the European Reference Network for Rare Neurological Diseases (ERN-RND) collaborated with the European Molecular Genetics Quality Network (EMQN) to establish an external quality assessment scheme for NGS-based diagnostics in RNDs. The scheme, initiated in 2021 with a pilot involving 29 labs and followed by a second round in 2022 with 42 labs, aimed to evaluate the performance of laboratories in genetic testing for RNDs. Each participating lab analysed genetic data from three hypothetical cases, assessing genotyping, interpretation, and clerical accuracy. Despite a majority of labs using exome or genome sequencing, there was considerable variability in gene content, sequencing quality, adherence to standards, and clinical guidance provision. Results showed that while most labs provided correct molecular diagnoses, there was significant variability in reporting technical quality, adherence to interpretation standards, reporting strategies, and clinical commentary. Notably, some labs returned results with the potential for adverse medical outcomes. This underscores the need for further harmonisation, guideline development, and external quality assessment in the evolving landscape of genomic diagnostics for RNDs. Overall, the experience with the scheme highlighted the generally good quality of participating labs but emphasised the imperative for ongoing improvement in data analysis, interpretation, and reporting to enhance patient safety.

The role of cochlear and vestibular afferents in long-latency cervical vestibular evoked myogenic potentials.

OBJECTIVE: To examine the origin of cervical vestibular evoked myogenic potential (cVEMP) late waves (n34-p44) elicited with air-conducted click stimuli. DESIGN: Using a retrospective design, cVEMPs from normal volunteers were compared to those obtained from patients with vestibular and auditory pathologies. STUDY SAMPLE: (1) Normal volunteers (n = 56); (2) severe-to-profound sensorineural hearing loss (SNHL) with normal vestibular function (n = 21); (3) peripheral vestibular impairment with preserved hearing (n = 16); (4) total vestibulocochlear deficit (n = 23). RESULTS: All normal volunteers had ipsilateral-dominant early p13-n23 peaks. Late peaks were present bilaterally in 78%. The p13-n23 response was present in all patients with SNHL but normal vestibular function, and 43% had late waves. Statistical comparison of these patients to a subset of age-matched controls showed no significant difference in the frequencies, amplitudes or latencies of their ipsilateral early and late peaks. cVEMPs were absent in all patients with vestibular impairment. CONCLUSION: The presence of long-latency cVEMP waves was not dependent on the integrity of sensorineural hearing pathways, but instead correlated with intact vestibular function. This finding conflicts with the view that these late waves are cochlear in origin, and suggests that vestibular afferents may assume a more prominent role in their generation.

Biallelic variants in Plexin B2 (PLXNB2) cause amelogenesis imperfecta, hearing loss and intellectual disability.

BACKGROUND: Plexins are large transmembrane receptors for the semaphorin family of signalling proteins. Semaphorin-plexin signalling controls cellular interactions that are critical during development as well as in adult life stages. Nine plexin genes have been identified in humans, but despite the apparent importance of plexins in development, only biallelic PLXND1 and PLXNA1 variants have so far been associated with Mendelian genetic disease. METHODS: Eight individuals from six families presented with a recessively inherited variable clinical condition, with core features of amelogenesis imperfecta (AI) and sensorineural hearing loss (SNHL), with variable intellectual disability. Probands were investigated by exome or genome sequencing. Common variants and those unlikely to affect function were excluded. Variants consistent with autosomal recessive inheritance were prioritised. Variant segregation analysis was performed by Sanger sequencing. RNA expression analysis was conducted in C57Bl6 mice. RESULTS: Rare biallelic pathogenic variants in plexin B2 (PLXNB2), a large transmembrane semaphorin receptor protein, were found to segregate with disease in all six families. The variants identified include missense, nonsense, splicing changes and a multiexon deletion. Plxnb2 expression was detected in differentiating ameloblasts. CONCLUSION: We identify rare biallelic pathogenic variants in PLXNB2 as a cause of a new autosomal recessive, phenotypically diverse syndrome with AI and SNHL as core features. Intellectual disability, ocular disease, ear developmental abnormalities and lymphoedema were also present in multiple cases. The variable syndromic human phenotype overlaps with that seen in Plxnb2 knockout mice, and, together with the rarity of human PLXNB2 variants, may explain why pathogenic variants in PLXNB2 have not been reported previously.

Bi-allelic mutation of CTNNB1 causes a severe form of syndromic microphthalmia, persistent foetal vasculature and vitreoretinal dysplasia.

BACKGROUND: Inherited vitreoretinopathies arise as a consequence of congenital retinal vascularisation abnormalities. They represent a phenotypically and genetically heterogeneous group of disorders that can have a major impact on vision. Several genes encoding proteins and effectors of the canonical Wnt/ß-catenin pathway have been associated and precise diagnosis, although difficult, is essential for proper clinical management including syndrome specific management where appropriate. This work aimed to investigate the molecular basis of disease in a single proband born to consanguineous parents, who presented with microphthalmia, persistent foetal vasculature, posterior lens vacuoles, vitreoretinal dysplasia, microcephaly, hypotelorism and global developmental delay, and was registered severely visually impaired by 5 months of age. METHODS: Extensive genomic pre-screening, including microarray comparative genomic hybridisation and sequencing of a 114 gene panel associated with cataract and congenital ophthalmic disorders was conducted by an accredited clinical laboratory. Whole exome sequencing (WES) was undertaken on a research basis and in vitro TOPflash transcriptional reporter assay was utilised to assess the impact of the putative causal variant. RESULTS: In the proband, WES revealed a novel, likely pathogenic homozygous mutation in the cadherin-associated protein beta-1 gene (CTNNB1), c.884C>G; p.(Ala295Gly), which encodes a co-effector molecule of the Wnt/ß-catenin pathway. The proband's parents were shown to be heterozygous carriers but ophthalmic examination did not detect any abnormalities. Functional assessment of the missense variant demonstrated significant reduction of ß-catenin activity. CONCLUSIONS: This is the first report of a biallelic disease-causing variation in CTNNB1. We conclude that this biallelic, transcriptional inactivating mutation of CTNNB1 causes a severe, syndromic form of microphthalmia, persistent foetal vasculature and vitreoretinal dysplasia that results in serious visual loss in infancy.

New variants and in silico analyses in GRK1 associated Oguchi disease.

Biallelic mutations in G-Protein coupled receptor kinase 1 (GRK1) cause Oguchi disease, a rare subtype of congenital stationary night blindness (CSNB). The purpose of this study was to identify disease causing GRK1 variants and use in-depth bioinformatic analyses to evaluate how their impact on protein structure could lead to pathogenicity. Patients' genomic DNA was sequenced by whole genome, whole exome or focused exome sequencing. Disease associated variants, published and novel, were compared to nondisease associated missense variants. The impact of GRK1 missense variants at the protein level were then predicted using a series of computational tools. We identified twelve previously unpublished cases with biallelic disease associated GRK1 variants, including eight novel variants, and reviewed all GRK1 disease associated variants. Further structure-based scoring revealed a hotspot for missense variants in the kinase domain. In addition, to aid future clinical interpretation, we identified the bioinformatics tools best able to differentiate disease associated from nondisease associated variants. We identified GRK1 variants in Oguchi disease patients and investigated how disease-causing variants may impede protein function in-silico.

Craniosynostosis-microphthalmia syndrome belongs to the spectrum of BCOR-related disorders.

Craniosynostosis-microphthalmia linked to BCOR haploinsufficiency.

Small Molecules Restore Bestrophin 1 Expression and Function of Both Dominant and Recessive Bestrophinopathies in Patient-Derived Retinal Pigment Epithelium.

Purpose: Bestrophinopathies are a group of untreatable inherited retinal dystrophies caused by mutations in the retinal pigment epithelium (RPE) Cl- channel bestrophin 1. We tested whether sodium phenylbutyrate (4PBA) could rescue the function of mutant bestrophin 1 associated with autosomal dominant and recessive disease. We then sought analogues of 4PBA with increased potency and determined the mode of action for 4PBA and a lead compound 2-naphthoxyacetic acid (2-NOAA). Lastly, we tested if 4PBA and 2-NOAA could functionally rescue bestrophin 1 function in RPE generated from induced pluripotent stem cells (iPSC-RPEs) derived from patients with a dominant or recessive bestrophinopathy. Methods: Global and plasma membrane expression was determined by Western blot and immunofluorescent microscopy, respectively. The effect of 4PBA and 2-NOAA on transcription was measured by quantitative RT-PCR and the rate of protein turnover by cycloheximide chase and Western blot. Channel function was measured by whole-cell patch clamp. Results: 4PBA and 2-NOAA can rescue the global and membrane expression of mutant bestrophin 1 associated with autosomal dominant disease (Best vitelliform macular dystrophy [BVMD]) and autosome recessive bestrophinopathy (ARB), and these small molecules have different modes of action. Both 4PBA and 2-NOAA significantly increased the channel function of mutant BVMD and ARB bestrophin 1 in HEK293T and iPSC-RPE cells derived from patients with BVMD and ARB. For 4PBA, the increased mutant channel function in BVMD and ARB iPSC-RPE was equal to that of wild-type iPSC-RPE bestrophin 1. Conclusions: The restoration of bestrophin 1 function in patient-derived RPE confirms the US Food and Drug Administration-approved drug 4PBA as a promising therapeutic treatment for bestrophinopathies.

Using an integrative machine learning approach utilising homology modelling to clinically interpret genetic variants: CACNA1F as an exemplar.

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.

Diagnostic yield of panel-based genetic testing in syndromic inherited retinal disease.

Thirty percent of all inherited retinal disease (IRD) is accounted for by conditions with extra-ocular features. This study aimed to establish the genetic diagnostic pick-up rate for IRD patients with one or more extra-ocular features undergoing panel-based screening in a clinical setting. One hundred and six participants, tested on a gene panel which contained both isolated and syndromic IRD genes, were retrospectively ascertained from the Manchester Genomic Diagnostics Laboratory database spanning 6 years (2012-2017). Phenotypic features were extracted from the clinical notes and classified according to Human Phenotype Ontology; all identified genetic variants were interpreted in accordance to the American College of Medical Genetics and Genomics guidelines. Overall, 49% (n = 52) of patients received a probable genetic diagnosis. A further 6% (n = 6) had a single disease-associated variant in an autosomal recessive disease-relevant gene. Fifty-two percent (n = 55) of patients had a clinical diagnosis at the time of testing. Of these, 71% (n = 39) received a probable genetic diagnosis. By contrast, for those without a provisional clinical diagnosis (n = 51), only 25% (n = 13) received a probable genetic diagnosis. The clinical diagnosis of Usher (n = 33) and Bardet-Biedl syndrome (n = 10) was confirmed in 67% (n = 22) and 80% (n = 8), respectively. The testing diagnostic rate in patients with clinically diagnosed multisystemic IRD conditions was significantly higher than those without one (71% versus 25%; p value < 0.001). The lower pick-up rate in patients without a clinical diagnosis suggests that panel-based approaches are unlikely to be the most effective means of achieving a molecular diagnosis for this group. Here, we suggest that genome-wide approaches (whole exome or genome) are more appropriate.

Clinical utility of genetic testing in 201 preschool children with inherited eye disorders.

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.

Loss-of-Function Mutations in the CFH Gene Affecting Alternatively Encoded Factor H-like 1 Protein Cause Dominant Early-Onset Macular Drusen.

PURPOSE: To characterize the molecular mechanism underpinning early-onset macular drusen (EOMD), a phenotypically severe subtype of age-related macular degeneration (AMD), in a subgroup of patients. DESIGN: Multicenter case series, in vitro experimentation, and retrospective analysis of previously reported variants. PARTICIPANTS: Seven families with apparently autosomal dominant EOMD. METHODS: Patients underwent a comprehensive ophthalmic assessment. Affected individuals from families A, B, and E underwent whole exome sequencing. The probands from families C, D, F, and G underwent Sanger sequencing analysis of the complement factor H (CFH) gene. Mutant recombinant factor H like-1 (FHL-1) proteins were expressed in HEK293 cells to assess the impact on FHL-1 expression and function. Previously reported EOMD-causing variants in CFH were reviewed. MAIN OUTCOME MEASURES: Detailed clinical phenotypes, genomic findings, in vitro characterization of mutation effect on protein function, and postulation of the pathomechanism underpinning EOMD. RESULTS: All affected participants demonstrated bilateral drusen. The earliest reported age of onset was 16 years (median, 46 years). Ultra-rare (minor allele frequency [MAF], ≤0.0001) CFH variants were identified as the cause of disease in each family: CFH c.1243del, p.(Ala415ProfsTer39) het; c.350+1G→T het; c.619+1G→A het, c.380G→A, p.(Arg127His) het; c.694C→T p.(Arg232Ter) het (identified in 2 unrelated families in this cohort); and c.1291T→A, p.(Cys431Ser). All mutations affect complement control protein domains 2 through 7, and thus are predicted to impact both FHL-1, the predominant isoform in Bruch's membrane (BrM) of the macula, and factor H (FH). In vitro analysis of recombinant proteins FHL-1R127H, FHL-1A415f/s, and FHL-1C431S demonstrated that they are not secreted, and thus are loss-of-function proteins. Review of 29 previously reported EOMD-causing mutations found that 75.8% (22/29) impact FHL-1 and FH. In total, 86.2% (25/29) of EOMD-associated variants cause haploinsufficiency of FH or FHL-1. CONCLUSIONS: Early-onset macular drusen is an underrecognized, phenotypically severe subtype of AMD. We propose that haploinsufficiency of FHL-1, the main regulator of the complement pathway in BrM, where drusen develop, is an important mechanism underpinning the development of EOMD in a number of cases. Understanding the molecular basis of EOMD will shed light on AMD pathogenesis given their pathologic similarities.

GLS hyperactivity causes glutamate excess, infantile cataract and profound developmental delay.

Loss-of-function mutations in glutaminase (GLS), the enzyme converting glutamine into glutamate, and the counteracting enzyme glutamine synthetase (GS) cause disturbed glutamate homeostasis and severe neonatal encephalopathy. We report a de novo Ser482Cys gain-of-function variant in GLS encoding GLS associated with profound developmental delay and infantile cataract. Functional analysis demonstrated that this variant causes hyperactivity and compensatory downregulation of GLS expression combined with upregulation of the counteracting enzyme GS, supporting pathogenicity. Ser482Cys-GLS likely improves the electrostatic environment of the GLS catalytic site, thereby intrinsically inducing hyperactivity. Alignment of +/-12.000 GLS protein sequences from >1000 genera revealed extreme conservation of Ser482 to the same degree as catalytic residues. Together with the hyperactivity, this indicates that Ser482 is evolutionarily preserved to achieve optimal-but submaximal-GLS activity. In line with GLS hyperactivity, increased glutamate and decreased glutamine concentrations were measured in urine and fibroblasts. In the brain (both grey and white matter), glutamate was also extremely high and glutamine was almost undetectable, demonstrated with magnetic resonance spectroscopic imaging at clinical field strength and subsequently supported at ultra-high field strength. Considering the neurotoxicity of glutamate when present in excess, the strikingly high glutamate concentrations measured in the brain provide an explanation for the developmental delay. Cataract, a known consequence of oxidative stress, was evoked in zebrafish expressing the hypermorphic Ser482Cys-GLS and could be alleviated by inhibition of GLS. The capacity to detoxify reactive oxygen species was reduced upon Ser482Cys-GLS expression, providing an explanation for cataract formation. In conclusion, we describe an inborn error of glutamate metabolism caused by a GLS hyperactivity variant, illustrating the importance of balanced GLS activity.

Bi-allelic Loss-of-Function Variants in DNMBP Cause Infantile Cataracts.

Infantile and childhood-onset cataracts form a heterogeneous group of disorders; among the many genetic causes, numerous pathogenic variants in additional genes associated with autosomal-recessive infantile cataracts remain to be discovered. We identified three consanguineous families affected by bilateral infantile cataracts. Using exome sequencing, we found homozygous loss-of-function variants in DNMBP: nonsense variant c.811C>T (p.Arg271∗) in large family F385 (nine affected individuals; LOD score = 5.18 at θ = 0), frameshift deletion c.2947_2948del (p.Asp983∗) in family F372 (two affected individuals), and frameshift variant c.2852_2855del (p.Thr951Metfs∗41) in family F3 (one affected individual). The phenotypes of all affected individuals include infantile-onset cataracts. RNAi-mediated knockdown of the Drosophila ortholog still life (sif), enriched in lens-secreting cells, affects the development of these cells as well as the localization of E-cadherin, alters the distribution of septate junctions in adjacent cone cells, and leads to a ∼50% reduction in electroretinography amplitudes in young flies. DNMBP regulates the shape of tight junctions, which correspond to the septate junctions in invertebrates, as well as the assembly pattern of E-cadherin in human epithelial cells. E-cadherin has an important role in lens vesicle separation and lens epithelial cell survival in humans. We therefore conclude that DNMBP loss-of-function variants cause infantile-onset cataracts in humans.

De Novo and Inherited Loss-of-Function Variants in TLK2: Clinical and Genotype-Phenotype Evaluation of a Distinct Neurodevelopmental Disorder.

Next-generation sequencing is a powerful tool for the discovery of genes related to neurodevelopmental disorders (NDDs). Here, we report the identification of a distinct syndrome due to de novo or inherited heterozygous mutations in Tousled-like kinase 2 (TLK2) in 38 unrelated individuals and two affected mothers, using whole-exome and whole-genome sequencing technologies, matchmaker databases, and international collaborations. Affected individuals had a consistent phenotype, characterized by mild-borderline neurodevelopmental delay (86%), behavioral disorders (68%), severe gastro-intestinal problems (63%), and facial dysmorphism including blepharophimosis (82%), telecanthus (74%), prominent nasal bridge (68%), broad nasal tip (66%), thin vermilion of the upper lip (62%), and upslanting palpebral fissures (55%). Analysis of cell lines from three affected individuals showed that mutations act through a loss-of-function mechanism in at least two case subjects. Genotype-phenotype analysis and comparison of computationally modeled faces showed that phenotypes of these and other individuals with loss-of-function variants significantly overlapped with phenotypes of individuals with other variant types (missense and C-terminal truncating). This suggests that haploinsufficiency of TLK2 is the most likely underlying disease mechanism, leading to a consistent neurodevelopmental phenotype. This work illustrates the power of international data sharing, by the identification of 40 individuals from 26 different centers in 7 different countries, allowing the identification, clinical delineation, and genotype-phenotype evaluation of a distinct NDD caused by mutations in TLK2.

Assessment of the incorporation of CNV surveillance into gene panel next-generation sequencing testing for inherited retinal diseases.

BACKGROUND: Diagnostic use of gene panel next-generation sequencing (NGS) techniques is commonplace for individuals with inherited retinal dystrophies (IRDs), a highly genetically heterogeneous group of disorders. However, these techniques have often failed to capture the complete spectrum of genomic variation causing IRD, including CNVs. This study assessed the applicability of introducing CNV surveillance into first-tier diagnostic gene panel NGS services for IRD. METHODS: Three read-depth algorithms were applied to gene panel NGS data sets for 550 referred individuals, and informatics strategies used for quality assurance and CNV filtering. CNV events were confirmed and reported to referring clinicians through an accredited diagnostic laboratory. RESULTS: We confirmed the presence of 33 deletions and 11 duplications, determining these findings to contribute to the confirmed or provisional molecular diagnosis of IRD for 25 individuals. We show that at least 7% of individuals referred for diagnostic testing for IRD have a CNV within genes relevant to their clinical diagnosis, and determined a positive predictive value of 79% for the employed CNV filtering techniques. CONCLUSION: Incorporation of CNV analysis increases diagnostic yield of gene panel NGS diagnostic tests for IRD, increases clarity in diagnostic reporting and expands the spectrum of known disease-causing mutations.

Validation of copy number variation analysis for next-generation sequencing diagnostics.

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.

Association of Steroid 5α-Reductase Type 3 Congenital Disorder of Glycosylation With Early-Onset Retinal Dystrophy.

Importance: Steroid 5α-reductase type 3 congenital disorder of glycosylation (SRD5A3-CDG) is a rare disorder of N-linked glycosylation. Its retinal phenotype is not well described but could be important for disease recognition because it appears to be a consistent primary presenting feature. Objective: To investigate a series of patients with the same mutation in the SRD5A3 gene and thereby characterize its retinal manifestations and other associated features. Design, Setting and Participants: Seven affected individuals from 4 unrelated families with early-onset retinal dystrophy as a primary manifestation underwent comprehensive ophthalmic assessment, including retinal imaging and electrodiagnostic testing. Developmental and systemic findings were also recorded. Molecular genetic approaches, including targeted next-generation sequencing, autozygosity mapping, and apex microarray, were tried to reach a diagnosis; all participants were mutation negative. Whole-exome sequencing or whole-genome sequencing was used to identify the causative variant. Biochemical profiling was conducted to confirm a CDG type I defect. Patient phenotype data were collected over the course of ophthalmic follow-up, spanning a period of 20 years, beginning March 20, 1997, through September 15, 2016. Main Outcomes and Measures: Detailed clinical phenotypes as well as genetic and biochemical results. Results: The cohort consisted of 7 participants (5 females and 2 males) whose mean (SD) age at the most recent examination was 17.1 (3.9) years and who were all of South Asian ethnicity. Whole-exome sequencing and whole-genome sequencing identified the same homozygous SRD5A3 c.57G>A, p.(Trp19Ter) variant as the underlying cause of early-onset retinal dystrophy in each family. Detailed ocular phenotyping identified early-onset (aged ≤3 years) visual loss (mean [SD] best-corrected visual acuity, +0.95 [0.34] logMAR [20/180 Snellen]), childhood-onset nyctalopia, myopia (mean [SD] refractive error, -6.71 [-4.22]), and nystagmus. Six of the 7 patients had learning difficulties and psychomotor delay. Fundus autofluorescence imaging and optical coherence tomographic scans were abnormal in all patients, and electrodiagnostic testing revealed rod and cone dysfunction in the 5 patients tested. Conclusions and Relevance: Mutations in the SRD5A3 gene may cause early-onset retinal dystrophy, a previously underdescribed feature of the SRD5A3-CDG disorder that is progressive and may lead to serious visual impairment. SRD5A3 and other glycosylation disorder genes should be considered as a cause of retinal dystrophy even when systemic features are mild. Further delineation of SRD5A3-associated eye phenotypes can help inform genetic counseling for prognostic estimation of visual loss and disease progression.

Panel-Based Clinical Genetic Testing in 85 Children with Inherited Retinal Disease.

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.