The landscape of submicroscopic structural variants at the OPN1LW/OPN1MW gene cluster on Xq28 underlying blue cone monochromacy.

Blue cone monochromacy (BCM) is an X-linked retinal disorder characterized by low vision, photoaversion, and poor color discrimination. BCM is due to the lack of long-wavelength-sensitive and middle-wavelength-sensitive cone photoreceptor function and caused by mutations in the OPN1LW/OPN1MW gene cluster on Xq28. Here, we investigated the prevalence and the landscape of submicroscopic structural variants (SVs) at single-base resolution in BCM patients. We found that about one-third (n = 73) of the 213 molecularly confirmed BCM families carry an SV, most commonly deletions restricted to the OPN1LW/OPN1MW gene cluster. The structure and precise breakpoints of the SVs were resolved in all but one of the 73 families. Twenty-two families-all from the United States-showed the same SV, and we confirmed a common ancestry of this mutation. In total, 42 distinct SVs were identified, including 40 previously unreported SVs, thereby quadrupling the number of precisely mapped SVs underlying BCM. Notably, there was no "region of overlap" among these SVs. However, 90% of SVs encompass the upstream locus control region, an essential enhancer element. Its minimal functional extent based on deletion mapping in patients was refined to 358 bp. Breakpoint analyses suggest diverse mechanisms underlying SV formation as well as in one case the gene conversion-based exchange of a 142-bp deletion between opsin genes. Using parsimonious assumptions, we reconstructed the composition and copy number of the OPN1LW/OPN1MW gene cluster prior to the mutation event and found evidence that large gene arrays may be predisposed to the occurrence of SVs at this locus.

Single Center Experience with Voretigene Neparvovec Gene Augmentation Therapy in RPE65 Mutation-Associated Inherited Retinal Degeneration in a Clinical Setting.

PURPOSE: To assess the impact of baseline data on psychophysical and morphological outcomes of subretinal voretigene neparvovec (VN) (Luxturna, Spark Therapeutics, Inc.) treatment. DESIGN: Single-center, retrospective, longitudinal, consecutive case series. PARTICIPANTS: Patients with RPE65-biallelic mutation-associated inherited retinal degeneration (RPE65-IRD) treated between February 2020 and March 2022 with VN and oral immunosuppression according to the manufacturer's recommendation by one surgeon (F.G.H.). METHODS: Retrospective analysis of surgical and clinical records, ancillary testing, and retinal imaging after VN therapy for RPE65-IRD. Descriptive statistics compared data at baseline up to 32 months post-treatment. MAIN OUTCOME MEASURES: Best-corrected visual acuity (BCVA), low-luminance VA (LLVA), Goldmann visual fields (GVFs), chromatic full-field stimulus threshold (FST) testing (FST), scotopic and photopic 2-color threshold perimetry (2CTP), and multimodal retinal imaging. RESULTS: Thirty eyes of 19 patients were analyzed (10 pediatric patients < 20 years; 20 adult patients > 20 years of age; overall range: 8-40 years) with a median follow-up of 15 months (range, 1-32). The fovea was completely or partially detached in 16 eyes, attached in 12 eyes, and not assessable in 2 eyes on intraoperative imaging. Median BCVA at baseline was better in the pediatric group (P < 0.05) and did not change significantly independent of age. Meaningful loss of BCVA (≥ 0.3 logarithm of the minimal angle of resolution [logMAR]) occurred in 5 of 18 adult eyes, and a meaningful gain (≥-0.3 logMAR) occurred in 2 of 18 adult and 2 of 8 pediatric eyes. The LLVA and scotopic 2CTP improved considerably in pediatric patients. Scotopic blue FST improved at all ages but more in pediatric patients (8/8 eyes gained ≥ 10 decibels [dB]; P < 0.05). In pediatric patients, median GVF improved by 20% for target V4e and by 50% for target III4e (target I4e not detected). Novel atrophy developed in 13 of 26 eyes at the site of the bleb or peripheral of vascular arcades. Improvements in FST did not correlate with development of chorioretinal atrophy at 12 months. Mean central retinal thickness was 165.87 µm (± 26.26) at baseline (30 eyes) and 157.69 µm (± 30.3) at 12 months (26 eyes). Eight adult patients were treated unilaterally. The untreated eyes did not show meaningful changes during follow-up. CONCLUSIONS: These data in a clinical setting show the effectiveness of VN therapy with stable median BCVA and mean retinal thickness and improvements of LLVA, FST, and 2CTP up to 32 months. Treatment effects were superior in the pediatric group. We observed new chorioretinal atrophy in 50% of the treated eyes. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

Biallelic mutations in L-dopachrome tautomerase (DCT) cause infantile nystagmus and oculocutaneous albinism.

Infantile nystagmus syndrome (INS) denominates early-onset, involuntary oscillatory eye movements with different etiologies. Nystagmus is also one of the symptoms in oculocutaneus albinism (OCA), a heterogeneous disease mainly caused by defects in melanin synthesis or melanosome biogenesis. Dopachrome tautomerase (DCT, also called TYRP2) together with tyrosinase (TYR) and tyrosin-related protein 1 (TYRP1) is one of the key enzymes in melanin synthesis. Although DCT´s role in pigmentation has been proven in different species, until now only mutations in TYR and TYRP1 have been found in patients with OCA. Detailed ophthalmological and orthoptic investigations identified a consanguineous family with two individuals with isolated infantile nystagmus and one family member with subtle signs of albinism. By whole-exome sequencing and segregation analysis, we identified the missense mutation c.176G > T (p.Gly59Val) in DCT in a homozygous state in all three affected family members. We show that this mutation results in incomplete protein maturation and targeting in vitro compatible with a partial or total loss of function. Subsequent screening of a cohort of patients with OCA (n = 85) and INS (n = 25) revealed two heterozygous truncating mutations, namely c.876C > A (p.Tyr292*) and c.1407G > A (p.Trp469*), in an independent patient with OCA. Taken together, our data suggest that mutations in DCT can cause a phenotypic spectrum ranging from isolated infantile nystagmus to oculocutaneous albinism.

Biallelic mutation of human SLC6A6 encoding the taurine transporter TAUT is linked to early retinal degeneration.

We previously reported that inactivation of the transmembrane taurine transporter (TauT or solute carrier 6a6) causes early retinal degeneration in mice. Compatible with taurine's indispensability for cell volume homeostasis, protein stabilization, cytoprotection, antioxidation, and immuno- and neuromodulation, mice develop multisystemic dysfunctions (hearing loss; liver fibrosis; and behavioral, heart, and skeletal muscle abnormalities) later on. Here, by genetic, cell biologic, in vivo1H-magnetic resonance spectroscopy and molecular dynamics simulation studies, we conducted in-depth characterization of a novel disorder: human TAUT deficiency. Loss of TAUT function due to a homozygous missense mutation caused panretinal degeneration in 2 brothers. TAUTp.A78E still localized in the plasma membrane but is predicted to impact structural stabilization. 3H-taurine uptake by peripheral blood mononuclear cells was reduced by 95%, and taurine levels were severely reduced in plasma, skeletal muscle, and brain. Extraocular dysfunctions were not yet detected, but significantly increased urinary excretion of 8-oxo-7,8-dihydroguanosine indicated generally enhanced (yet clinically unapparent) oxidative stress and RNA oxidation, warranting continuous broad surveillance.-Preising, M. N., Görg, B., Friedburg, C., Qvartskhava, N., Budde, B. S., Bonus, M., Toliat, M. R., Pfleger, C., Altmüller, J., Herebian, D., Beyer, M., Zöllner, H. J., Wittsack, H.-J., Schaper, J., Klee, D., Zechner, U., Nürnberg, P., Schipper, J., Schnitzler, A., Gohlke, H., Lorenz, B., Häussinger, D., Bolz, H. J. Biallelic mutation of human SLC6A6 encoding the taurine transporter TAUT is linked to early retinal degeneration.

Where are the missing gene defects in inherited retinal disorders? Intronic and synonymous variants contribute at least to 4% of CACNA1F-mediated inherited retinal disorders.

Inherited retinal disorders (IRD) represent clinically and genetically heterogeneous diseases. To date, pathogenic variants have been identified in ~260 genes. Albeit that many genes are implicated in IRD, for 30-50% of the cases, the gene defect is unknown. These cases may be explained by novel gene defects, by overlooked structural variants, by variants in intronic, promoter or more distant regulatory regions, and represent synonymous variants of known genes contributing to the dysfunction of the respective proteins. Patients with one subgroup of IRD, namely incomplete congenital stationary night blindness (icCSNB), show a very specific phenotype. The major cause of this condition is the presence of a hemizygous pathogenic variant in CACNA1F. A comprehensive study applying direct Sanger sequencing of the gene-coding regions, exome and genome sequencing applied to a large cohort of patients with a clinical diagnosis of icCSNB revealed indeed that seven of the 189 CACNA1F-related cases have intronic and synonymous disease-causing variants leading to missplicing as validated by minigene approaches. These findings highlight that gene-locus sequencing may be a very efficient method in detecting disease-causing variants in clinically well-characterized patients with a diagnosis of IRD, like icCSNB.

Splitting of the lateral rectus muscle with medial transposition to treat oculomotor palsy: a retrospective analysis of 29 consecutive cases.

PURPOSE: The lateralis splitting technique has been an interesting option for treating large-angle exotropia due to complete 3rd nerve paralysis since its inception in the early 1990s. The purpose of this study is to report on our experience regarding the effectiveness and complications of this method. METHODS: Retrospective analysis of a consecutive series of 29 patients operated by one single experienced surgeon and examined according to a specific operative and perioperative protocol. Patients were examined preoperatively, on the 2nd day and 3rd month after surgery. Outcome measures include strabismus angle, horizontal motility, head turn, binocular function, and incidence and resolution of postoperative serous retinal detachment as seen with infrared imaging and spectral domain optical coherence tomography (SD-OCT). RESULTS: Surgery brought about a large and stable reduction of strabismus angle and head turn. It reduced horizontal motility, but moved the range of monocular excursion much closer to center. Eighty percent of patients with constant diplopia acquired some fields of single binocular vision. A significant number of cases (33.3%) developed transitory serous retinal detachment with varying onset and extent. CONCLUSION: This is by far the largest published study regarding the outcome of lateralis splitting in NIII palsy. The procedure is difficult, yet a very useful option. Serous detachment is a serious complication, but usually transitory. Its cause and mechanisms are not fully understood and warrant further investigation.

[The Phenotypic Spectrum of Ophthalmic Changes in CEP290 Mutations]. / Das phänotypische Spektrum der Augenveränderungen bei CEP290-Sequenzvariationen.

PURPOSE: Inherited retinal diseases (IRDs) may be caused by variations in genes affecting the connecting cilium of photoreceptor cells and intraflagellar transport, manifested as ciliopathies. CEP290 is frequently mutated in non-syndromic, but also syndromic IRDs. In preparation for clinical treatment trials, detailed phenotypic work-up including longitudinal follow-up is mandatory. METHODS: We performed genotype-phenotype correlations in 30 patients with biallelic mutations in CEP290. The study was approved by the IRB of the medical faculty of the Justus-Liebig University Giessen. The patients received a comprehensive clinical examination, including spectral-domain optical coherence tomography (SD-OCT), fundus autofluorescence (FAF) recording, and electrophysiology whenever possible. RESULTS: Thirty patients aged 1 month to 84 years (median at first visit 0.6 y) were followed between 5 months and 25.7 years (median 4.8 y). Twenty-three of these patients carried the c.2991+1655A>G mutation (30/60 allele 2, 7 homozygous). The second most frequent mutation was p.K1575* (9/60 alleles). The full-field electroretinogram showed residual response in a few patients only. After progression, electrophysiological responses were below threshold in all patients. Severely reduced visual acuity persisted from birth. Eight patients had quantifiable best corrected visual acuity (BCVA, logMAR 2 - 0.3), in one case up to the age of 84 y. Absent fixation of targets was noted in 15 patients during the first months of life. Ten of these patients did not improve during follow-up past the second year of life. The other patients developed at least light perception (LP, n = 7) or hand movement (HM, n = 3). Better BCVA was not restricted to the c.2991+1655A>G mutation. Fundus photography documented degenerative changes throughout the retina with macular degeneration and circular increased fundus autofluorescence signals (9/30 patients) in the perimacular ring and in the rod ring, and spotty changes in the periphery. SD-OCT (6/30 patients) disclosed reduced photoreceptor layer (OPL to OS) thickness and preserved inner retinal thickness (RNFL to INL). Better BCVA did not correlate to genotype or central photoreceptor layer thickness. CONCLUSION: As reported earlier, CEP290 variations are one of the most frequent causes of IRDs with infancy onset. In our patient cohort of 30 patients, only 33% had no LP, 67% at least LP, and among these 26% logMAR 2 to 0.3. Together with preserved ganglion cell and nerve fibre cell layers, success with gene therapeutic approaches appears possible.

Recessive Mutations in RTN4IP1 Cause Isolated and Syndromic Optic Neuropathies.

Autosomal-recessive optic neuropathies are rare blinding conditions related to retinal ganglion cell (RGC) and optic-nerve degeneration, for which only mutations in TMEM126A and ACO2 are known. In four families with early-onset recessive optic neuropathy, we identified mutations in RTN4IP1, which encodes a mitochondrial ubiquinol oxydo-reductase. RTN4IP1 is a partner of RTN4 (also known as NOGO), and its ortholog Rad8 in C. elegans is involved in UV light response. Analysis of fibroblasts from affected individuals with a RTN4IP1 mutation showed loss of the altered protein, a deficit of mitochondrial respiratory complex I and IV activities, and increased susceptibility to UV light. Silencing of RTN4IP1 altered the number and morphogenesis of mouse RGC dendrites in vitro and the eye size, neuro-retinal development, and swimming behavior in zebrafish in vivo. Altogether, these data point to a pathophysiological mechanism responsible for RGC early degeneration and optic neuropathy and linking RTN4IP1 functions to mitochondrial physiology, response to UV light, and dendrite growth during eye maturation.

[Overview of Congenital Stationary Night Blindness with Predominantly Normal Fundus Appearance]. / Überblick über die kongenitale stationäre Nachtblindheit mit überwiegend normalem Fundus.

Congenital stationary night blindness (CSNB) is a clinically and genetically heterogeneous group of non-progressive retinal disorder with largely normal fundus appearance. The mode of inheritance can be autosomal dominant (adCSNB), autosomal recessive (arCSNB) or X-chromosomal (XLCSNB). Additional ocular signs can be myopia, hyperopia, strabismus, nystagmus and reduced visual acuity. The Riggs and Schubert-Bornschein form of CSNB can be discriminated by electroretinography. While the Riggs form represents a dysfunction of the rods, a signal transmission defect from photoreceptors to bipolar cell is described in patients with the more frequently occurring Schubert-Bornschein form. The Schubert-Bornschein form can be further divided into incomplete (icCSNB) and complete (cCSNB) showing different electroretinograms (ERGs). While patients with cCSNB show a dysfunction of the ON-signaling pathway, patients with icCSNB show a dysfunction of the ON- and OFF-signaling pathways, affecting visual acuity as well. Using classical linkage, candidate gene analyses and more recent next-generation sequencing approaches, to date, mutations in 13 different genes have been associated with this disease. In vitro and in vivo models showed a correlation of the phenotype of patients with the expression, protein localization and function of the respective molecules: genes, mutated in patients with the Riggs form of CSNB have an important role in the rod phototransduction cascade. Genes mutated in patients with icCSNB, code for proteins important for glutamate neurotransmitter release at the synaptic cleft of the photoreceptors. Genes mutated in patients with cCSNB, code for proteins important for glutamate uptake and further signal transmission to the ON-bipolar cells. Preliminary in vivo studies showed that CSNB may be cured by gene therapy. These studies concerning CSNB are important for the precise diagnosis of patients with this disease, but are also helpful in deciphering key molecules essential for signal transmission from photoreceptors to bipolar cells. So far, it is a poorly understood field.

Spatially Resolved Spectral Sensitivities as a Potential Read-out Parameter in Clinical Gene Therapeutic Trials.

PURPOSE: Spatially resolved functional assessment of rods and cones under photopic and scotopic conditions is desirable to evaluate the treatment outcome of gene therapeutic applications in inherited retinal disorders, such as early- onset severe retinal dystrophy (EOSRD) or achromatopsia. METHODS: A sample of 3 healthy subjects, 6 patients with RPE65 deficiency (aged 11-45 years), and 3 patients with cone dysfunction disorders underwent spectral sensitivity testing (SST) under conditions of dark and light adaptation using a Humphrey Field Analyzer modified perimeter. RESULTS: SST in healthy subjects revealed sensitivity curves corresponding well with the CIE (International Commission on Illumination) standard fundamentals. Absence of cone function was observed in patients with cone dysfunction disorders. In patients with RPE65 mutations, SST under conditions of both dark and light adaptation revealed similar curves at typical cone sensitivities. S cone-related thresholds were diminished in young patients (11-14 years) and absent in adults (19 years and over). CONCLUSION: In the present study, residual vision was cone mediated both under photopic and scotopic conditions in young patients with EOSRD associated with RPE65 mutations, but S cone function was severely reduced early on. In rod monochromats, vision was rod mediated both under conditions of dark and light adaptation. These observations are important for ongoing and future clinical trials employing gene therapeutic strategies in both rod-cone dystrophies and achromatopsia.

Mosaic synaptopathy and functional defects in Cav1.4 heterozygous mice and human carriers of CSNB2.

Mutations in CACNA1F encoding the α1-subunit of the retinal Cav1.4 L-type calcium channel have been linked to Cav1.4 channelopathies including incomplete congenital stationary night blindness type 2A (CSNB2), Åland Island eye disease (AIED) and cone-rod dystrophy type 3 (CORDX3). Since CACNA1F is located on the X chromosome, Cav1.4 channelopathies are typically affecting male patients via X-chromosomal recessive inheritance. Occasionally, clinical symptoms have been observed in female carriers, too. It is currently unknown how these mutations lead to symptoms in carriers and how the retinal network in these females is affected. To investigate these clinically important issues, we compared retinal phenotypes in Cav1.4-deficient and Cav1.4 heterozygous mice and in human female carrier patients. Heterozygous Cacna1f carrier mice have a retinal mosaic consistent with differential X-chromosomal inactivation, characterized by adjacent vertical columns of affected and non-affected wild-type-like retinal network. Vertical columns in heterozygous mice are well comparable to either the wild-type retinal network of normal mice or to the retina of homozygous mice. Affected retinal columns display pronounced rod and cone photoreceptor synaptopathy and cone degeneration. These changes lead to vastly impaired vision-guided navigation under dark and normal light conditions and reduced retinal electroretinography (ERG) responses in Cacna1f carrier mice. Similar abnormal ERG responses were found in five human CACNA1F carriers, four of which had novel mutations. In conclusion, our data on Cav1.4 deficient mice and human female carriers of mutations in CACNA1F are consistent with a phenotype of mosaic CSNB2.

Whole-exome sequencing identifies mutations in GPR179 leading to autosomal-recessive complete congenital stationary night blindness.

Congenital stationary night blindness (CSNB) is a heterogeneous retinal disorder characterized by visual impairment under low light conditions. This disorder is due to a signal transmission defect from rod photoreceptors to adjacent bipolar cells in the retina. Two forms can be distinguished clinically, complete CSNB (cCSNB) or incomplete CSNB; the two forms are distinguished on the basis of the affected signaling pathway. Mutations in NYX, GRM6, and TRPM1, expressed in the outer plexiform layer (OPL) lead to disruption of the ON-bipolar cell response and have been seen in patients with cCSNB. Whole-exome sequencing in cCSNB patients lacking mutations in the known genes led to the identification of a homozygous missense mutation (c.1807C>T [p.His603Tyr]) in one consanguineous autosomal-recessive cCSNB family and a homozygous frameshift mutation in GPR179 (c.278delC [p.Pro93Glnfs(∗)57]) in a simplex male cCSNB patient. Additional screening with Sanger sequencing of 40 patients identified three other cCSNB patients harboring additional allelic mutations in GPR179. Although, immunhistological studies revealed Gpr179 in the OPL in wild-type mouse retina, Gpr179 did not colocalize with specific ON-bipolar markers. Interestingly, Gpr179 was highly concentrated in horizontal cells and Müller cell endfeet. The involvement of these cells in cCSNB and the specific function of GPR179 remain to be elucidated.

Shared decision-making, control preferences and psychological well-being in patients with RPE65 deficiency awaiting experimental gene therapy.

PURPOSE: Retinal gene therapy trials are currently ongoing in a small number of inherited retinal disorders and this number is expected to rise significantly. The aim of this study was to analyze the psychological aspects of patients with RPE65 deficiency awaiting potential enrollment in gene therapy trials. METHODS: Five patients with genetically proven RPE65 deficiency took part in this study. They were asked to complete the German versions of (i) the Patient Health Questionnaire (PHQ-D), (ii) the National Eye Institute Visual Function Questionnaire (NEI-VFQ), (iii) the Shared Decision Making Questionnaire (PEF-FB-9), and (iv) the Autonomy Preference Index (API-Dm), and in addition they took part in qualitative interviews. RESULTS: The evaluations of the questionnaires and the interviews showed that the patients have quite high information needs and wish to take part in medical decisions. The perspective to participate in gene therapy trials does not seem to cause pronounced worries. Only the insecurity about if and when enrollment in a trial takes place may be burdensome. DISCUSSION: This study generated important data about the psychological situation of patients awaiting potential enrollment in clinical trials, which can be used to improve patient care in the increasing number of future gene therapy trials around the world.

Normative values of peripheral retinal thickness measured with Spectralis OCT in healthy young adults.

BACKGROUND: To evaluate the range of peripheral retinal thickness (PRT) in young healthy human subjects by Spectralis HRA + OCT, and to analyze a potential association between the peripheral location, spherical equivalent (SE), axial length (AL), and gender. METHODS: After pupil dilation, the peripheral retina was scanned by means of six volume protocols (9 × 7.5 mm), each consisting of 31 B-scans. PRT was determined at 4,500, 5,500, 6,500 and 7,500 µm eccentricity from the fovea and the optic nerve head (ONH). Data points were collected every 22.5°. Six additional data points at a distance of 9,000 µm were included. In 11 subjects, OCT measurements were performed twice to evaluate reproducibility. Coefficients of variation (COV) were calculated. RESULTS: Randomly selected eyes of 50 subjects (19-30 years) with AL of 21-27 mm (SE: -5.75 to +5.25 dpt) were included in the study. Mean PRT decreased significantly (p ≤ 0.001, r = -0.99) towards the periphery, reaching a minimum at 9,000 µm eccentricity (mean PRT: 187.7 ± 8.9 µm). Multiple regression analysis revealed a significant association of PRT with AL at nasal and temporal locations as well as gender for temporal locations. COVs ranged from 0.44 to 2.90 %, with highest COVs found nasal to the fovea. CONCLUSIONS: This is the first study to report normative data of PRT outside the ETDRS grid and to show a significant continuous almost linear decrease of the RT from the center into the periphery. The data will be valuable to detect peripheral pathologies of the retina in early stages of peripheral retinal dystrophies.

Screening of a large cohort of leber congenital amaurosis and retinitis pigmentosa patients identifies novel LCA5 mutations and new genotype-phenotype correlations.

This study was undertaken to investigate the prevalence of sequence variants in LCA5 in patients with Leber congenital amaurosis (LCA), early-onset retinal dystrophy (EORD), and autosomal recessive retinitis pigmentosa (arRP); to delineate the ocular phenotypes; and to provide an overview of all published LCA5 variants in an online database. Patients underwent standard ophthalmic evaluations after providing informed consent. In selected patients, optical coherence tomography (OCT) and fundus autofluorescence imaging were possible. DNA samples from 797 unrelated patients with LCA and 211 with the various types of retinitis pigmentosa (RP) were screened by Sanger sequence analysis of all LCA5 exons and intron/exon junctions. Some LCA patients were prescreened by APEX technology or selected based on homozygosity mapping. In silico analyses were performed to assess the pathogenicity of the variants. Segregation analysis was performed where possible. Published and novel LCA5 variants were collected, amended for their correct nomenclature, and listed in a Leiden Open Variation Database (LOVD). Sequence analysis identified 18 new probands with 19 different LCA5 variants. Seventeen of the 19 LCA5 variants were novel. Except for two missense variants and one splice site variant, all variants were protein-truncating mutations. Most patients expressed a severe phenotype, typical of LCA. However, some LCA subjects had better vision and intact inner segment/outer segment (IS/OS) junctions on OCT imaging. In two families with LCA5 variants, the phenotype was more compatible with EORD with affected individuals displaying preserved islands of retinal pigment epithelium. One of the families with a milder phenotype harbored a homozygous splice site mutation; a second family was found to have a combination of a stop mutation and a missense mutation. This is the largest LCA5 study to date. We sequenced 1,008 patients (797 with LCA, 211 with arRP) and identified 18 probands with LCA5 mutations. Mutations in LCA5 are a rare cause of childhood retinal dystrophy accounting for ∼2% of disease in this cohort, and the majority of LCA5 mutations are likely null. The LCA5 protein truncating mutations are predominantly associated with LCA. However, in two families with the milder EORD, the LCA5 gene analysis revealed a homozygous splice site mutation in one and a stop mutation in combination with a missense mutation in a second family, suggesting that this milder phenotype is due to residual function of lebercilin and expanding the currently known phenotypic spectrum to include the milder early onset RP. Some patients have remaining foveal cone structures (intact IS/OS junctions on OCT imaging) and remaining visual acuities, which may bode well for upcoming treatment trials.

TRPM1 is mutated in patients with autosomal-recessive complete congenital stationary night blindness.

Night vision requires signaling from rod photoreceptors to adjacent bipolar cells in the retina. Mutations in the genes NYX and GRM6, expressed in ON bipolar cells, lead to a disruption of the ON bipolar cell response. This dysfunction is present in patients with complete X-linked and autosomal-recessive congenital stationary night blindness (CSNB) and can be assessed by standard full-field electroretinography (ERG), showing severely reduced rod b-wave amplitude and slightly altered cone responses. Although many cases of complete CSNB (cCSNB) are caused by mutations in NYX and GRM6, in approximately 60% of the patients the gene defect remains unknown. Animal models of human diseases are a good source for candidate genes, and we noted that a cCSNB phenotype present in homozygous Appaloosa horses is associated with downregulation of TRPM1. TRPM1, belonging to the family of transient receptor potential channels, is expressed in ON bipolar cells and therefore qualifies as an excellent candidate. Indeed, mutation analysis of 38 patients with CSNB identified ten unrelated cCSNB patients with 14 different mutations in this gene. The mutation spectrum comprises missense, splice-site, deletion, and nonsense mutations. We propose that the cCSNB phenotype in these patients is due to the absence of functional TRPM1 in retinal ON bipolar cells.

Screening of TYR, OCA2, GPR143, and MC1R in patients with congenital nystagmus, macular hypoplasia, and fundus hypopigmentation indicating albinism.

BACKGROUND: A broad spectrum of pigmentation of the skin and hair is found among patients diagnosed with ocular albinism (OA) and oculocutaneous albinism (OCA). Even though complexion is variable, three ocular features, i.e., hypopigmentation of the fundus, hypoplasia of the macula, and nystagmus, are classical pathological findings in these patients. We screened 172 index patients with a clinical diagnosis of OA or OCA based on the classical findings, to evaluate the frequency of sequence variants in tyrosinase (TYR), P-gene, P-protein (OCA2), and the G-protein-coupled receptor 143 gene, OA1 (GPR143). In addition, we investigated the association of sequence variants in the melanocortin receptor 1 gene (MC1R) and OCA2. METHODS: Pigmentation of the hair, skin, iris, and fundus were included in the evaluation of OCA and OA. Male OA patients showing X-linked inheritance were screened for GPR143. Females showing OA without family history were regarded as representing autosomal recessive OA (OA3). Direct sequencing was applied to PCR products showing aberrant single-strand conformation polymorphism-banding patterns. RESULTS: Fifty-seven male index patients were screened for OA. We identified 16 potentially pathogenic sequence variations in GPR143 (10 novel) in 22 males. In TYR, we identified 23 (7 novel), and in OCA2 28 (11 novel) possibly pathogenic variants. Variants on both alleles were identified in TYR or OCA2 in 29/79 OCA patients and 14/71 OA patients. Sequence changes in TYR were identified almost exclusively in OCA patients, while sequence changes in OCA2 occurred in OCA and OA patients. MC1R sequencing was performed in 47 patients carrying mutations in OCA2 and revealed MC1R mutations in 42 of them. CONCLUSIONS: TYR gene mutations have a more severe effect on pigmentation than mutations in OCA2 and the GPR143 gene. Nevertheless, mutations in these genes affect the development of visual function either directly or by interaction with other genes like MC1R, which can be deduced from a frequent association of MC1R variants with p.R305W or p.R419Q in OCA2.

Fundus albipunctatus associated with compound heterozygous mutations in RPE65.

PURPOSE: To describe a family with an 18-year-old woman with fundus albipunctatus and compound heterozygous mutations in RPE65 whose unaffected parents and 1 female sibling harbored single heterozygous RPE65 mutations. DESIGN: Observational study. PARTICIPANTS: Four family members. METHODS: Clinical examinations included full-field electroretinogram (ffERG) after standard (30-minute) and prolonged (17-hour) dark adaptation, multifocal electroretinogram (mfERG), optical coherence tomography (OCT), and fundus autofluorescence (FAF). Molecular genetic testing included sequencing of RDH5 and RLBP1 and screening for known autosomal-recessive retinitis pigmentosa mutations by a commercially available microarray technique. RPE65 sequencing was performed after the identification of a known heterozygous splice-site mutation by array screening. MAIN OUTCOME MEASURES: We recorded ffERG and mfERG amplitudes, OCT characteristics, the FAF intensity index, and the outcomes of DNA sequencing regarding RPE65 mutations. RESULTS: Uniform, yellow-white dots typical of fundus albipunctatus were demonstrated in the proband. These dots corresponded with discrete, hyperreflective formations extending from the Bruch's membrane and retinal pigment epithelium (RPE) into the level of the external limiting membrane, thus spanning along the entire RPE and photoreceptor outer and inner segments. A reduced thickness of the central retina and the RPE-outer segment complex was demonstrated. The intensity of the FAF was severely reduced in the entire fundus. At age 18, ffERG-including prolonged dark adaptation-demonstrated a barely recordable rod response after standard dark adaptation and normalization (increase by more than 700%) of the response after prolonged dark adaptation. The cone 30-Hz flicker response was reduced after standard dark adaptation and increased by >50% after prolonged dark adaptation. In addition, mfERG demonstrated reduced central and peripheral responses. Molecular genetic analysis demonstrated compound heterozygous mutations (IVS1+5G>A and c.344T>C) in RPE65. No mutations were found in RDH5 or RLBP1. No significant abnormalities of retinal structure or function were detected in the parents and sister carrying single heterozygous mutations in RPE65. CONCLUSIONS: This is the first reported association between compound heterozygous RPE65 mutations and fundus albipunctatus, indicative of a mutation-specific phenotypic effect in this gene. This finding, together with the reduced FAF, supports that disruption of retinoid recycling in the RPE is essential for the development of fundus albipunctatus.