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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.

Next-generation sequencing has revolutionized rare disease diagnostics, but many patients remain without a molecular diagnosis, particularly because many candidate variants usually survive despite strict filtering. Exomiser was launched in 2014 as a Java tool that performs an integrative analysis of patients' sequencing data and their phenotypes encoded with Human Phenotype Ontology (HPO) terms. It prioritizes variants by leveraging information on variant frequency, predicted pathogenicity, and gene-phenotype associations derived from human diseases, model organisms, and protein-protein interactions. Early published releases of Exomiser were able to prioritize disease-causative variants as top candidates in up to 97% of simulated whole-exomes. The size of the tested real patient datasets published so far are very limited. Here, we present the latest Exomiser version 12.0.1 with many new features. We assessed the performance using a set of 134 whole-exomes from patients with a range of rare retinal diseases and known molecular diagnosis. Using default settings, Exomiser ranked the correct diagnosed variants as the top candidate in 74% of the dataset and top 5 in 94%; not using the patients' HPO profiles (i.e., variant-only analysis) decreased the performance to 3% and 27%, respectively. In conclusion, Exomiser is an effective support tool for rare Mendelian phenotype-driven variant prioritization.

In a subset of inherited retinal degenerations (including cone, cone-rod, and macular dystrophies), cone photoreceptors are more severely affected than rods; ABCA4 mutations are the most common cause of this heterogeneous class of disorders. To identify retinal-disease-associated genes, we performed exome sequencing in 28 individuals with "cone-first" retinal disease and clinical features atypical for ABCA4 retinopathy. We then conducted a gene-based case-control association study with an internal exome data set as the control group. TTLL5, encoding a tubulin glutamylase, was highlighted as the most likely disease-associated gene; 2 of 28 affected subjects harbored presumed loss-of-function variants: c.[1586_1589delAGAG];[1586_1589delAGAG], p.[Glu529Valfs(∗)2];[Glu529Valfs(∗)2], and c.[401delT(;)3354G>A], p.[Leu134Argfs(∗)45(;)Trp1118(∗)]. We then inspected previously collected exome sequence data from individuals with related phenotypes and found two siblings with homozygous nonsense variant c.1627G>T (p.Glu543(∗)) in TTLL5. Subsequently, we tested a panel of 55 probands with retinal dystrophy for TTLL5 mutations; one proband had a homozygous missense change (c.1627G>A [p.Glu543Lys]). The retinal phenotype was highly similar in three of four families; the sibling pair had a more severe, early-onset disease. In human and murine retinae, TTLL5 localized to the centrioles at the base of the connecting cilium. TTLL5 has been previously reported to be essential for the correct function of sperm flagella in mice and play a role in polyglutamylation of primary cilia in vitro. Notably, genes involved in the polyglutamylation and deglutamylation of tubulin have been associated with photoreceptor degeneration in mice. The electrophysiological and fundus autofluorescence imaging presented here should facilitate the molecular diagnosis in further families.

PurposeTo characterize retinal structure and function in achromatopsia (ACHM) in preparation for clinical trials of gene therapy.DesignCross-sectional study.ParticipantsForty subjects with ACHM.MethodsAll subjects underwent spectral domain optical coherence tomography (SD-OCT), microperimetry, and molecular genetic testing. Foveal structure on SD-OCT was graded into 5 distinct categories: (1) continuous inner segment ellipsoid (ISe), (2) ISe disruption, (3) ISe absence, (4) presence of a hyporeflective zone (HRZ), and (5) outer retinal atrophy including retinal pigment epithelial loss. Foveal and outer nuclear layer (ONL) thickness was measured and presence of hypoplasia determined.Main outcome measuresPhotoreceptor appearance on SD-OCT imaging, foveal and ONL thickness, presence of foveal hypoplasia, retinal sensitivity and fixation stability, and association of these parameters with age and genotype.ResultsForty subjects with a mean age of 24.9 years (range, 6-52 years) were included. Disease-causing variants were found in CNGA3 (n = 18), CNGB3 (n = 15), GNAT2 (n = 4), and PDE6C (n = 1). No variants were found in 2 individuals. In all, 22.5% of subjects had a continuous ISe layer at the fovea, 27.5% had ISe disruption, 20% had an absent ISe layer, 22.5% had an HRZ, and 7.5% had outer retinal atrophy. No significant differences in age (P = 0.77), mean retinal sensitivity (P = 0.21), or fixation stability (P = 0.34) across the 5 SD-OCT categories were evident. No correlation was found between age and foveal thickness (P = 0.84) or between age and foveal ONL thickness (P = 0.12).ConclusionsThe lack of a clear association of disruption of retinal structure or function in ACHM with age suggests that the window of opportunity for intervention by gene therapy is wider in some individuals than previously indicated. Therefore, the potential benefit for a given subject is likely to be better predicted by specific measurement of photoreceptor structure rather than simply by age. The ability to directly assess cone photoreceptor preservation with SD-OCT and/or adaptive optics imaging is likely to prove invaluable in selecting subjects for future trials and measuring the trials' impact.

Foveal hypoplasia and optic nerve misrouting are developmental defects of the visual pathway and only co-occur in connection with albinism; to date, they have only been associated with defects in the melanin-biosynthesis pathway. Here, we report that these defects can occur independently of albinism in people with recessive mutations in the putative glutamine transporter gene SLC38A8. Nine different mutations were identified in seven Asian and European families. Using morpholino-mediated ablation of Slc38a8 in medaka fish, we confirmed that pigmentation is unaffected by loss of SLC38A8. Furthermore, by undertaking an association study with SNPs at the SLC38A8 locus, we showed that common variants within this gene modestly affect foveal thickness in the general population. This study reveals a melanin-independent component underpinning the development of the visual pathway that requires a functional role for SLC38A8.

PurposeTo examine retinal structure and changes in photoreceptor intensity after dark adaptation in patients with complete congenital stationary night blindness and Oguchi disease.DesignProspective, observational case series.MethodsWe recruited 3 patients with complete congenital stationary night blindness caused by mutations in GRM6, 2 brothers with Oguchi disease caused by mutations in GRK1, and 1 normal control. Retinal thickness was measured from optical coherence tomography images. Integrity of the rod and cone mosaic was assessed using adaptive optics scanning light ophthalmoscopy. We imaged 5 of the patients after a period of dark adaptation and examined layer reflectivity on optical coherence tomography in a patient with Oguchi disease under light- and dark-adapted conditions.ResultsRetinal thickness was reduced in the parafoveal region in patients with GRM6 mutations as a result of decreased thickness of the inner retinal layers. All patients had normal photoreceptor density at all locations analyzed. On removal from dark adaptation, the intensity of the rods (but not cones) in the patients with Oguchi disease gradually and significantly increased. In 1 Oguchi disease patient, the outer segment layer contrast on optical coherence tomography was 4-fold higher under dark-adapted versus light-adapted conditions.ConclusionsThe selective thinning of the inner retinal layers in patients with GRM6 mutations suggests either reduced bipolar or ganglion cell numbers or altered synaptic structure in the inner retina. Our finding that rods, but not cones, change intensity after dark adaptation suggests that fundus changes in Oguchi disease are the result of changes within the rods as opposed to changes at a different retinal locus.

PurposeTo report the clinical phenotype in patients with a retinal dystrophy associated with novel mutations in the MER tyrosine kinase (MERTK) gene.MethodsA consanguineous family of Middle Eastern origin was identified, and affected members underwent a full clinical evaluation. Linkage analysis was performed using the Affymetrix 50K chip. Regions of homozygosity were identified. The positional candidate genes protocadherin 21 (PCDH21), retinal G protein-coupled receptor (RGR), and MERTK were polymerase chain reaction (PCR) amplified and sequenced. Long-range PCR was performed to characterize the deletion. Two hundred and ninety-two probands with autosomal recessive, childhood onset, retinal dystrophies were analyzed using the Asper Ophthalmics Leber congenital amaurosis chip to screen for known MERTK mutations.ResultsAnalysis of a 50K-Affymetrix whole genome scan identified three regions of homozygosity on chromosomes 2 and 10. Screening of the candidate gene MERTK showed a possible deletion of exon 8. Long-range PCR identified a ~9 kb deletion within MERTK that removes exon 8. Screening of DNA from a panel of Saudi Arabian patients with autosomal recessive retinitis pigmentosa identified a second consanguineous family with the same mutation. One patient with a known MERTK mutation (p.R651X) was identified using the Asper Ophthalmics Leber congenital amaurosis chip. Further screening of the gene identified a second novel splice site mutation in intron 1. The phenotype associated with these identified MERTK mutations is of a childhood onset rod-cone dystrophy with early macular atrophy. The optical coherence tomography (OCT) appearance is distinctive with evidence of debris beneath the sensory retina.ConclusionsMutations in MERTK are a rare cause of retinal dystrophy. Non homologous recombination between Alu Y repeats near or within disease genes may be an important cause of retinal dystrophies.