Multidisciplinary team directed analysis of whole genome sequencing reveals pathogenic non-coding variants in molecularly undiagnosed inherited retinal dystrophies.

The purpose of this paper is to identify likely pathogenic non-coding variants in inherited retinal dystrophy (IRD) genes, using genome sequencing (GS). Patients with IRD were recruited to the study and underwent comprehensive ophthalmological evaluation and GS. The results of GS were investigated through virtual gene panel analysis, and plausible pathogenic variants and clinical phenotype evaluated by the multidisciplinary team (MDT) discussion. For unsolved patients in whom a specific gene was suspected to harbor a missed pathogenic variant, targeted re-analysis of non-coding regions was performed on GS data. Candidate variants were functionally tested by messenger RNA analysis, minigene or luciferase reporter assays. Previously unreported, likely pathogenic, non-coding variants in 7 genes (PRPF31, NDP, IFT140, CRB1, USH2A, BBS10 and GUCY2D), were identified in 11 patients. These were shown to lead to mis-splicing (PRPF31, IFT140, CRB1 and USH2A) or altered transcription levels (BBS10 and GUCY2D). MDT-led, phenotype-driven, non-coding variant re-analysis of GS is effective in identifying the missing causative alleles.

Neuropathy target esterase activity defines phenotypes among PNPLA6 disorders.

Biallelic pathogenic variants in the PNPLA6 gene cause a broad spectrum of disorders leading to gait disturbance, visual impairment, anterior hypopituitarism and hair anomalies. PNPLA6 encodes neuropathy target esterase (NTE), yet the role of NTE dysfunction on affected tissues in the large spectrum of associated disease remains unclear. We present a systematic evidence-based review of a novel cohort of 23 new patients along with 95 reported individuals with PNPLA6 variants that implicate missense variants as a driver of disease pathogenesis. Measuring esterase activity of 46 disease-associated and 20 common variants observed across PNPLA6-associated clinical diagnoses unambiguously reclassified 36 variants as pathogenic and 10 variants as likely pathogenic, establishing a robust functional assay for classifying PNPLA6 variants of unknown significance. Estimating the overall NTE activity of affected individuals revealed a striking inverse relationship between NTE activity and the presence of retinopathy and endocrinopathy. This phenomenon was recaptured in vivo in an allelic mouse series, where a similar NTE threshold for retinopathy exists. Thus, PNPLA6 disorders, previously considered allelic, are a continuous spectrum of pleiotropic phenotypes defined by an NTE genotype:activity:phenotype relationship. This relationship, and the generation of a preclinical animal model, pave the way for therapeutic trials, using NTE as a biomarker.

Beyond factor H: The impact of genetic-risk variants for age-related macular degeneration on circulating factor-H-like 1 and factor-H-related protein concentrations.

Age-related macular degeneration (AMD) is a leading cause of vision loss; there is strong genetic susceptibility at the complement factor H (CFH) locus. This locus encodes a series of complement regulators: factor H (FH), a splice variant factor-H-like 1 (FHL-1), and five factor-H-related proteins (FHR-1 to FHR-5), all involved in the regulation of complement factor C3b turnover. Little is known about how AMD-associated variants at this locus might influence FHL-1 and FHR protein concentrations. We have used a bespoke targeted mass-spectrometry assay to measure the circulating concentrations of all seven complement regulators and demonstrated elevated concentrations in 352 advanced AMD-affected individuals for all FHR proteins (FHR-1, p = 2.4 × 10-10; FHR-2, p = 6.0 × 10-10; FHR-3, p = 1.5 × 10-5; FHR-4, p = 1.3 × 10-3; FHR-5, p = 1.9 × 10-4) and FHL-1 (p = 4.9 × 10-4) when these individuals were compared to 252 controls, whereas no difference was seen for FH (p = 0.94). Genome-wide association analyses in controls revealed genome-wide-significant signals at the CFH locus for all five FHR proteins, and univariate Mendelian-randomization analyses strongly supported the association of FHR-1, FHR-2, FHR-4, and FHR-5 with AMD susceptibility. These findings provide a strong biochemical explanation for how genetically driven alterations in circulating FHR proteins could be major drivers of AMD and highlight the need for research into FHR protein modulation as a viable therapeutic avenue for AMD.

Optogenetic approaches to therapy for inherited retinal degenerations.

Inherited retinal degenerations such as retinitis pigmentosa (RP) affect around one in 4000 people and are the leading cause of blindness in working age adults in several countries. In these typically monogenic conditions, there is progressive degeneration of photoreceptors; however, inner retinal neurons such as bipolar cells and ganglion cells remain largely structurally intact, even in end-stage disease. Therapeutic approaches aiming to stimulate these residual cells, independent of the underlying genetic cause, could potentially restore visual function in patients with advanced vision loss, and benefit many more patients than therapies directed at the specific gene implicated in each disorder. One approach investigated for this purpose is that of optogenetics, a method of neuromodulation that utilises light to activate neurons engineered to ectopically express a light-sensitive protein. Using gene therapy via adeno-associated viral vectors, a range of photosensitive proteins have been expressed in remaining retinal cells in advanced retinal degeneration with in vivo studies demonstrating restoration of visual function. Developing an effective optogenetic strategy requires consideration of multiple factors, including the light-sensitive protein that is used, the vector and method for gene delivery, and the target cell for expression because these in turn may affect the quality of vision that can be restored. Currently, at least four clinical trials are ongoing to investigate optogenetic therapies in patients, with the ultimate aim of reversing visual loss in end-stage disease.

Mutation in the intracellular chloride channel CLCC1 associated with autosomal recessive retinitis pigmentosa.

We identified a homozygous missense alteration (c.75C>A, p.D25E) in CLCC1, encoding a presumptive intracellular chloride channel highly expressed in the retina, associated with autosomal recessive retinitis pigmentosa (arRP) in eight consanguineous families of Pakistani descent. The p.D25E alteration decreased CLCC1 channel function accompanied by accumulation of mutant protein in granules within the ER lumen, while siRNA knockdown of CLCC1 mRNA induced apoptosis in cultured ARPE-19 cells. TALEN KO in zebrafish was lethal 11 days post fertilization. The depressed electroretinogram (ERG) cone response and cone spectral sensitivity of 5 dpf KO zebrafish and reduced eye size, retinal thickness, and expression of rod and cone opsins could be rescued by injection of wild type CLCC1 mRNA. Clcc1+/- KO mice showed decreased ERGs and photoreceptor number. Together these results strongly suggest that intracellular chloride transport by CLCC1 is a critical process in maintaining retinal integrity, and CLCC1 is crucial for survival and function of retinal cells.

Biallelic Mutation of ARHGEF18, Involved in the Determination of Epithelial Apicobasal Polarity, Causes Adult-Onset Retinal Degeneration.

Mutations in more than 250 genes are implicated in inherited retinal dystrophy; the encoded proteins are involved in a broad spectrum of pathways. The presence of unsolved families after highly parallel sequencing strategies suggests that further genes remain to be identified. Whole-exome and -genome sequencing studies employed here in large cohorts of affected individuals revealed biallelic mutations in ARHGEF18 in three such individuals. ARHGEF18 encodes ARHGEF18, a guanine nucleotide exchange factor that activates RHOA, a small GTPase protein that is a key component of tight junctions and adherens junctions. This biological pathway is known to be important for retinal development and function, as mutation of CRB1, encoding another component, causes retinal dystrophy. The retinal structure in individuals with ARHGEF18 mutations resembled that seen in subjects with CRB1 mutations. Five mutations were found on six alleles in the three individuals: c.808A>G (p.Thr270Ala), c.1617+5G>A (p.Asp540Glyfs∗63), c.1996C>T (p.Arg666∗), c.2632G>T (p.Glu878∗), and c.2738_2761del (p.Arg913_Glu920del). Functional tests suggest that each disease genotype might retain some ARHGEF18 activity, such that the phenotype described here is not the consequence of nullizygosity. In particular, the p.Thr270Ala missense variant affects a highly conserved residue in the DBL homology domain, which is required for the interaction and activation of RHOA. Previously, knock-out of Arhgef18 in the medaka fish has been shown to cause larval lethality which is preceded by retinal defects that resemble those seen in zebrafish Crumbs complex knock-outs. The findings described here emphasize the peculiar sensitivity of the retina to perturbations of this pathway, which is highlighted as a target for potential therapeutic strategies.

Comprehensive Rare Variant Analysis via Whole-Genome Sequencing to Determine the Molecular Pathology of Inherited Retinal Disease.

Inherited retinal disease is a common cause of visual impairment and represents a highly heterogeneous group of conditions. Here, we present findings from a cohort of 722 individuals with inherited retinal disease, who have had whole-genome sequencing (n = 605), whole-exome sequencing (n = 72), or both (n = 45) performed, as part of the NIHR-BioResource Rare Diseases research study. We identified pathogenic variants (single-nucleotide variants, indels, or structural variants) for 404/722 (56%) individuals. Whole-genome sequencing gives unprecedented power to detect three categories of pathogenic variants in particular: structural variants, variants in GC-rich regions, which have significantly improved coverage compared to whole-exome sequencing, and variants in non-coding regulatory regions. In addition to previously reported pathogenic regulatory variants, we have identified a previously unreported pathogenic intronic variant in CHM in two males with choroideremia. We have also identified 19 genes not previously known to be associated with inherited retinal disease, which harbor biallelic predicted protein-truncating variants in unsolved cases. Whole-genome sequencing is an increasingly important comprehensive method with which to investigate the genetic causes of inherited retinal disease.

Recurrent heterozygous PAX6 missense variants cause severe bilateral microphthalmia via predictable effects on DNA-protein interaction.

PURPOSE: Most classical aniridia is caused by PAX6 haploinsufficiency. PAX6 missense variants can be hypomorphic or mimic haploinsufficiency. We hypothesized that missense variants also cause previously undescribed disease by altering the affinity and/or specificity of PAX6 genomic interactions. METHODS: We screened PAX6 in 372 individuals with bilateral microphthalmia, anophthalmia, or coloboma (MAC) from the Medical Research Council Human Genetics Unit eye malformation cohort (HGUeye) and reviewed data from the Deciphering Developmental Disorders study. We performed cluster analysis on PAX6-associated ocular phenotypes by variant type and molecular modeling of the structural impact of 86 different PAX6 causative missense variants. RESULTS: Eight different PAX6 missense variants were identified in 17 individuals (15 families) with MAC, accounting for 4% (15/372) of our cohort. Seven altered the paired domain (p.[Arg26Gln]x1, p.[Gly36Val]x1, p.[Arg38Trp]x2, p.[Arg38Gln]x1, p.[Gly51Arg]x2, p.[Ser54Arg]x2, p.[Asn124Lys]x5) and one the homeodomain (p.[Asn260Tyr]x1). p.Ser54Arg and p.Asn124Lys were exclusively associated with severe bilateral microphthalmia. MAC-associated variants were predicted to alter but not ablate DNA interaction, consistent with the electrophoretic mobility shifts observed using mutant paired domains with well-characterized PAX6-binding sites. We found no strong evidence for novel PAX6-associated extraocular disease. CONCLUSION: Altering the affinity and specificity of PAX6-binding genome-wide provides a plausible mechanism for the worse-than-null effects of MAC-associated missense variants.

Genetic Basis of Inherited Retinal Disease in a Molecularly Characterized Cohort of More Than 3000 Families from the United Kingdom.

PURPOSE: In a large cohort of molecularly characterized inherited retinal disease (IRD) families, we investigated proportions with disease attributable to causative variants in each gene. DESIGN: Retrospective study of electronic patient records. PARTICIPANTS: Patients and relatives managed in the Genetics Service of Moorfields Eye Hospital in whom a molecular diagnosis had been identified. METHODS: Genetic screening used a combination of single-gene testing, gene panel testing, whole exome sequencing, and more recently, whole genome sequencing. For this study, genes listed in the Retinal Information Network online resource (https://sph.uth.edu/retnet/) were included. Transcript length was extracted for each gene (Ensembl, release 94). MAIN OUTCOME MEASURES: We calculated proportions of families with IRD attributable to variants in each gene in the entire cohort, a cohort younger than 18 years, and a current cohort (at least 1 patient encounter between January 1, 2017, and August 2, 2019). Additionally, we explored correlation between numbers of families and gene transcript length. RESULTS: We identified 3195 families with a molecular diagnosis (variants in 135 genes), including 4236 affected individuals. The pediatric cohort comprised 452 individuals from 411 families (66 genes). The current cohort comprised 2614 families (131 genes; 3130 affected individuals). The 20 most frequently implicated genes overall (with prevalence rates per families) were as follows: ABCA4 (20.8%), USH2A (9.1%), RPGR (5.1%), PRPH2 (4.6%), BEST1 (3.9%), RS1 (3.5%), RP1 (3.3%), RHO (3.3%), CHM (2.7%), CRB1 (2.1%), PRPF31 (1.8%), MY07A (1.7%), OPA1 (1.6%), CNGB3 (1.4%), RPE65 (1.2%), EYS (1.2%), GUCY2D (1.2%), PROM1 (1.2%), CNGA3 (1.1%), and RDH12 (1.1%). These accounted for 71.8% of all molecularly diagnosed families. Spearman coefficients for correlation between numbers of families and transcript length were 0.20 (P = 0.025) overall and 0.27 (P = 0.017), -0.17 (P = 0.46), and 0.71 (P = 0.047) for genes in which variants exclusively cause recessive, dominant, or X-linked disease, respectively. CONCLUSIONS: Our findings help to quantify the burden of IRD attributable to each gene. More than 70% of families showed pathogenic variants in 1 of 20 genes. Transcript length (relevant to gene delivery strategies) correlated significantly with numbers of affected families (but not for dominant disease).

Exome sequencing in patients with microphthalmia, anophthalmia, and coloboma (MAC) from a consanguineous population.

Next-generation sequencing strategies have resulted in mutation detection rates of 21% to 61% in small cohorts of patients with microphthalmia, anophthalmia and coloboma (MAC), but despite progress in identifying novel causative genes, many patients remain without a genetic diagnosis. We studied a cohort of 19 patients with MAC who were ascertained from a population with high rates of consanguinity. Using single nucleotide polymorphism (SNP) arrays and whole exome sequencing (WES), we identified one pathogenic variant in TENM3 in a patient with cataracts in addition to MAC. We also detected novel variants of unknown significance in genes that have previously been associated with MAC, including KIF26B, MICU1 and CDON, and identified variants in candidate genes for MAC from the Wnt signaling pathway, comprising LRP6, WNT2B and IQGAP1, but our findings do not prove causality. Plausible variants were not found for many of the cases, indicating that our current understanding of the pathogenesis of MAC, a highly heterogeneous group of ocular defects, remains incomplete.

SSBP1 mutations in dominant optic atrophy with variable retinal degeneration.

OBJECTIVE: Autosomal dominant optic atrophy (ADOA) starts in early childhood with loss of visual acuity and color vision deficits. OPA1 mutations are responsible for the majority of cases, but in a portion of patients with a clinical diagnosis of ADOA, the cause remains unknown. This study aimed to identify novel ADOA-associated genes and explore their causality. METHODS: Linkage analysis and sequencing were performed in multigeneration families and unrelated patients to identify disease-causing variants. Functional consequences were investigated in silico and confirmed experimentally using the zebrafish model. RESULTS: We defined a new ADOA locus on 7q33-q35 and identified 3 different missense variants in SSBP1 (NM_001256510.1; c.113G>A [p.(Arg38Gln)], c.320G>A [p.(Arg107Gln)] and c.422G>A [p.(Ser141Asn)]) in affected individuals from 2 families and 2 singletons with ADOA and variable retinal degeneration. The mutated arginine residues are part of a basic patch that is essential for single-strand DNA binding. The loss of a positive charge at these positions is very likely to lower the affinity of SSBP1 for single-strand DNA. Antisense-mediated knockdown of endogenous ssbp1 messenger RNA (mRNA) in zebrafish resulted in compromised differentiation of retinal ganglion cells. A similar effect was achieved when mutated mRNAs were administered. These findings point toward an essential role of ssbp1 in retinal development and the dominant-negative nature of the identified human variants, which is consistent with the segregation pattern observed in 2 multigeneration families studied. INTERPRETATION: SSBP1 is an essential protein for mitochondrial DNA replication and maintenance. Our data have established pathogenic variants in SSBP1 as a cause of ADOA and variable retinal degeneration. ANN NEUROL 2019;86:368-383.

Unique noncoding variants upstream of PRDM13 are associated with a spectrum of developmental retinal dystrophies including progressive bifocal chorioretinal atrophy.

The autosomal dominant progressive bifocal chorioretinal atrophy (PBCRA) disease locus has been mapped to chromosome 6q14-16.2 that overlaps the North Carolina macular dystrophy (NCMD) locus MCDR1. NCMD is a nonprogressive developmental macular dystrophy, in which variants upstream of PRDM13 have been implicated. Whole genome sequencing was performed to interrogate structural variants (SVs) and single nucleotide variants (SNVs) in eight individuals, six affected individuals from two families with PBCRA, and two individuals from an additional family with a related developmental macular dystrophy. A SNV (chr6:100,046,804T>C), located 7.8 kb upstream of the PRDM13 gene, was shared by all PBCRA-affected individuals in the disease locus. Haplotype analysis suggested that the variant arose independently in the two families. The two affected individuals from Family 3 were screened for rare variants in the PBCRA and NCMD loci. This revealed a de novo variant in the proband, 21 bp from the first SNV (chr6:100,046,783A>C). This study expands the noncoding variant spectrum upstream of PRDM13 and suggests altered spatio-temporal expression of PRDM13 as a candidate disease mechanism in the phenotypically distinct but related conditions, NCMD and PBCRA.

Mutations in CPAMD8 Cause a Unique Form of Autosomal-Recessive Anterior Segment Dysgenesis.

Anterior segment dysgeneses (ASDs) comprise a spectrum of developmental disorders affecting the anterior segment of the eye. Here, we describe three unrelated families affected by a previously unclassified form of ASD. Shared ocular manifestations include bilateral iris hypoplasia, ectopia lentis, corectopia, ectropion uveae, and cataracts. Whole-exome sequencing and targeted Sanger sequencing identified mutations in CPAMD8 (C3 and PZP-like alpha-2-macroglobulin domain-containing protein 8) as the cause of recessive ASD in all three families. A homozygous missense mutation in the evolutionarily conserved alpha-2-macroglobulin (A2M) domain of CPAMD8, c.4351T>C (p. Ser1451Pro), was identified in family 1. In family 2, compound heterozygous frameshift, c.2352_2353insC (p.Arg785Glnfs∗23), and splice-site, c.4549-1G>A, mutations were identified. Two affected siblings in the third family were compound heterozygous for splice-site mutations c.700+1G>T and c.4002+1G>A. CPAMD8 splice-site mutations caused aberrant pre-mRNA splicing in vivo or in vitro. Intriguingly, our phylogenetic analysis revealed rodent lineage-specific CPAMD8 deletion, precluding a developmental expression study in mice. We therefore investigated the spatiotemporal expression of CPAMD8 in the developing human eye. RT-PCR and in situ hybridization revealed CPAMD8 expression in the lens, iris, cornea, and retina early in development, including strong expression in the distal tips of the retinal neuroepithelium that form the iris and ciliary body, thus correlating CPAMD8 expression with the affected tissues. Our study delineates a unique form of recessive ASD and defines a role for CPAMD8, a protein of unknown function, in anterior segment development, implying another pathway for the pathogenicity of ASD.

Mutations in REEP6 Cause Autosomal-Recessive Retinitis Pigmentosa.

Retinitis pigmentosa (RP) is the most frequent form of inherited retinal dystrophy. RP is genetically heterogeneous and the genes identified to date encode proteins involved in a wide range of functional pathways, including photoreceptor development, phototransduction, the retinoid cycle, cilia, and outer segment development. Here we report the identification of biallelic mutations in Receptor Expression Enhancer Protein 6 (REEP6) in seven individuals with autosomal-recessive RP from five unrelated families. REEP6 is a member of the REEP/Yop1 family of proteins that influence the structure of the endoplasmic reticulum but is relatively unstudied. The six variants identified include three frameshift variants, two missense variants, and a genomic rearrangement that disrupts exon 1. Human 3D organoid optic cups were used to investigate REEP6 expression and confirmed the expression of a retina-specific isoform REEP6.1, which is specifically affected by one of the frameshift mutations. Expression of the two missense variants (c.383C>T [p.Pro128Leu] and c.404T>C [p.Leu135Pro]) and the REEP6.1 frameshift mutant in cultured cells suggest that these changes destabilize the protein. Furthermore, CRISPR-Cas9-mediated gene editing was used to produce Reep6 knock-in mice with the p.Leu135Pro RP-associated variant identified in one RP-affected individual. The homozygous knock-in mice mimic the clinical phenotypes of RP, including progressive photoreceptor degeneration and dysfunction of the rod photoreceptors. Therefore, our study implicates REEP6 in retinal homeostasis and highlights a pathway previously uncharacterized in retinal dystrophy.

PIGMENTED PARAVENOUS CHORIORETINAL ATROPHY: Detailed Clinical Study of a Large Cohort.

PURPOSE: To review and describe in detail the demographics, functional and anatomical characteristics, and clinical course of pigmented paravenous chorioretinal atrophy in a large cohort of adults and children. METHODS: This is a retrospective case series of consecutive patients diagnosed with pigmented paravenous chorioretinal atrophy at a single U.K. referral center from 1974 to 2016. Clinical records, retinal imaging (color fundus photography, fundus autofluorescence, and optical coherence tomography), and electrophysiological assessments were reviewed. RESULTS: Twenty-three patients were identified (13 males and 10 females). The mean age at presentation was 35 years (range 10-67 years). Mean follow-up was 6.7 years (range 0-30 years). There was no family history of similar retinal disease. Thirteen (57%) patients were asymptomatic. Symptoms included photopsia (n = 1.4%), blurred vision (n = 4.17%), peripheral visual field loss (n = 3.13%), and nyctalopia (n = 2.8%). One patient had previous intermediate uveitis. Twenty-one (91%) patients had ≥6/12 in the better seeing eye at final follow-up; visual acuity loss over time was recorded in 2 patients. Color vision was normal in all 14 patients assessed. Paravenous hypoautofluorescence with surrounding increased fundus autofluorescence was characteristically observed. Optical coherence tomography over the retinal changes demonstrated choroidal, retinal pigment epithelium, and outer retinal layer thinning. Peripapillary atrophic changes on fundus photography were evident in 20 (87%) patients. Interocular asymmetry of fundus and electroretinography findings was common. The electroretinography findings showed a similar degree of generalized rod and cone photoreceptor dysfunction in most cases. CONCLUSION: Overall, most patients with pigmented paravenous chorioretinal atrophy maintained stable vision. The lack of other affected family members, slow or absent progression, and interocular asymmetry of the retinal features is suggestive of an acquired rather than inherited retinal disorder, which is generally nonprogressive. We identify that patients commonly have marked interocular asymmetry both on structural and functional assessment.

Mutations in TUBGCP4 alter microtubule organization via the γ-tubulin ring complex in autosomal-recessive microcephaly with chorioretinopathy.

We have identified TUBGCP4 variants in individuals with autosomal-recessive microcephaly and chorioretinopathy. Whole-exome sequencing performed on one family with two affected siblings and independently on another family with one affected child revealed compound-heterozygous mutations in TUBGCP4. Subsequent Sanger sequencing was performed on a panel of individuals from 12 French families affected by microcephaly and ophthalmic manifestations, and one other individual was identified with compound-heterozygous mutations in TUBGCP4. One synonymous variant was common to all three families and was shown to induce exon skipping; the other mutations were frameshift mutations and a deletion. TUBGCP4 encodes γ-tubulin complex protein 4, a component belonging to the γ-tubulin ring complex (γ-TuRC) and known to regulate the nucleation and organization of microtubules. Functional analysis of individual fibroblasts disclosed reduced levels of the γ-TuRC, altered nucleation and organization of microtubules, abnormal nuclear shape, and aneuploidy. Moreover, zebrafish treated with morpholinos against tubgcp4 were found to have reduced head volume and eye developmental anomalies with chorioretinal dysplasia. In summary, the identification of TUBGCP4 mutations in individuals with microcephaly and a spectrum of anomalies in eye development, particularly photoreceptor anomalies, provides evidence of an important role for the γ-TuRC in brain and eye development.

Long-term effect of gene therapy on Leber's congenital amaurosis.

BACKGROUND: Mutations in RPE65 cause Leber's congenital amaurosis, a progressive retinal degenerative disease that severely impairs sight in children. Gene therapy can result in modest improvements in night vision, but knowledge of its efficacy in humans is limited. METHODS: We performed a phase 1-2 open-label trial involving 12 participants to evaluate the safety and efficacy of gene therapy with a recombinant adeno-associated virus 2/2 (rAAV2/2) vector carrying the RPE65 complementary DNA, and measured visual function over the course of 3 years. Four participants were administered a lower dose of the vector, and 8 were administered a higher dose. In a parallel study in dogs, we investigated the relationship among vector dose, visual function, and electroretinography (ERG) findings. RESULTS: Improvements in retinal sensitivity were evident, to varying extents, in six participants for up to 3 years, peaking at 6 to 12 months after treatment and then declining. No associated improvement in retinal function was detected by means of ERG. Three participants had intraocular inflammation, and two had clinically significant deterioration of visual acuity. The reduction in central retinal thickness varied among participants. In dogs, RPE65 gene therapy with the same vector at lower doses improved vision-guided behavior, but only higher doses resulted in improvements in retinal function that were detectable with the use of ERG. CONCLUSIONS: Gene therapy with rAAV2/2 RPE65 vector improved retinal sensitivity, albeit modestly and temporarily. Comparison with the results obtained in the dog model indicates that there is a species difference in the amount of RPE65 required to drive the visual cycle and that the demand for RPE65 in affected persons was not met to the extent required for a durable, robust effect. (Funded by the National Institute for Health Research and others; ClinicalTrials.gov number, NCT00643747.).

Leber Congenital Amaurosis Associated with Mutations in CEP290, Clinical Phenotype, and Natural History in Preparation for Trials of Novel Therapies.

PURPOSE: To investigate and describe in detail the demographics, functional and anatomic characteristics, and clinical course of Leber congenital amaurosis (LCA) associated with mutations in the CEP290 gene (LCA-CEP290) in a large cohort of adults and children. DESIGN: Retrospective case series. PARTICIPANTS: Patients with mutations in CEP290 identified at a single UK referral center. METHODS: Review of case notes and results of retinal imaging (color fundus photography, fundus autofluorescence [FAF] imaging, OCT), electrophysiologic assessment, and molecular genetic testing. MAIN OUTCOME MEASURES: Molecular genetic testing, clinical findings including visual acuity and retinal imaging, and electrophysiologic assessment. RESULTS: Forty patients with LCA-CEP290 were identified. The deep intronic mutation c.2991+1655 A>G was the most common disease-causing variant (23/40 patients) identified in the compound heterozygous state in 20 patients (50%) and homozygous in 2 patients (5%). Visual acuity (VA) varied from 6/9 to no perception of light, and only 2 of 12 patients with longitudinal VA data showed deterioration in VA in their better-seeing eye over time. A normal fundus was found at diagnosis in younger patients (mean age, 1.9 years), with older patients showing white flecks (mean age, 5.9 years) or pigmentary retinopathy (mean age, 21.7 years). Eleven of 12 patients (92%) with OCT imaging had preservation of foveal architecture. Ten of 12 patients (83%) with FAF imaging had a perifoveal hyperautofluorescent ring. Having 2 nonsense CEP290 mutations was associated with worse final VA and the presence of nonocular features. CONCLUSIONS: Detailed analysis of the clinical phenotype of LCA-CEP290 in a large cohort confirms that there is a window of opportunity in childhood for therapeutic intervention based on relative structural preservation in the central cone-rich retina in a significant proportion of patients, with the majority harboring the deep intronic variant potentially tractable to several planned gene editing approaches.

Early Patterns of Macular Degeneration in ABCA4-Associated Retinopathy.

PURPOSE: To describe the earliest features of ABCA4-associated retinopathy. DESIGN: Case series. PARTICIPANTS: Children with a clinical and molecular diagnosis of ABCA4-associated retinopathy without evidence of macular atrophy. METHODS: The retinal phenotype was characterized by color fundus photography, OCT, fundus autofluorescence (FAF) imaging, electroretinography, and in 2 patients, adaptive optics scanning laser ophthalmoscopy (AOSLO). Sequencing of the ABCA4 gene was performed in all patients. MAIN OUTCOME MEASURES: Visual acuity, OCT, FAF, electroretinography, and AOSLO results. RESULTS: Eight children with ABCA4-associated retinopathy without macular atrophy were identified. Biallelic variants in ABCA4 were identified in all patients. Four children were asymptomatic, and 4 reported loss of VA. Patients were young (median age, 8.5 years; interquartile range, 6.8 years) with good visual acuity (median, 0.155 logarithm of the minimum angle of resolution [logMAR]; interquartile range, 0.29 logMAR). At presentation, the macula appeared normal (n = 3), had a subtly altered foveal reflex (n = 4), or demonstrated manifest fine yellow dots (n = 1). Fundus autofluorescence identified hyperautofluorescent dots in the central macula in 3 patients, 2 of whom showed a normal fundus appearance. Only 1 child had widespread hyperautofluorescent retinal flecks at presentation. OCT imaging identified hyperreflectivity at the base of the outer nuclear layer in all 8 patients. Where loss of outer nuclear volume was evident, this appeared to occur preferentially at a perifoveal locus. Longitudinal split-detector AOSLO imaging in 2 individuals confirmed that the greatest change in cone spacing occurred in the perifoveal, and not foveolar, photoreceptors. Electroretinography showed a reduced B-wave-to-A-wave ratio in 3 of 5 patients tested; in 2 children, recordings clearly showed electronegative results. CONCLUSIONS: In childhood-onset ABCA4-associated retinopathy, the earliest stages of macular atrophy involve the parafovea and spare the foveola. In some cases, these changes are predated by tiny, foveal, yellow, hyperautofluorescent dots. Hyperreflectivity at the base of the outer nuclear layer, previously described as thickening of the external limiting membrane, is likely to represent a structural change at the level of the foveal cone nuclei. Electroretinography suggests that the initial site of retinal dysfunction may occur after phototransduction.

Congenital sodium diarrhea and chorioretinal coloboma with optic disc coloboma in a patient with biallelic SPINT2 mutations, including p.(Tyr163Cys).

Congenital sodium diarrhea is a rare and life-threatening disorder characterized by a severe, secretory diarrhea containing high concentrations of sodium, leading to hyponatremia and metabolic acidosis. It may occur in isolation or in association with systemic features such as facial dysmorphism, choanal atresia, imperforate anus, and corneal erosions. Mutations in the serine protease inhibitor, Kunitz-Type 2 (SPINT2) gene have been associated with congenital sodium diarrhea and additional syndromic features. We present a child with congenital sodium diarrhea, cleft lip and palate, corneal erosions, optic nerve coloboma, and intermittent exotropia who was found to have biallelic mutations in SPINT2. One mutation, c.488A > G, predicting p.(Tyr163Cys), has been previously associated with a syndromic form of congenital sodium diarrhea. The other mutation, c.166_167dupTA, predicting p.(Asn57Thrfs*24) has not previously been reported and is likely a novel pathogenic variant for this disorder. We found only one other report of an optic nerve coloboma associated with SPINT2 mutations and this occurred in a patient with congenital tufting enteropathy. Our patient confirms an association of ocular coloboma with presumed loss of SPINT2 function.