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.

Electrophysiology and glaucoma: current status and future challenges.

Visual electrophysiology allows non-invasive monitoring of the function of most processing stages along the visual pathway. Here, we consider which of the available methods provides the most information concerning glaucomatous optic nerve disease. The multifocal electroretinogram (ERG), although often employed, is less affected in glaucoma than two direct measurements of retinal ganglion cell function, namely the pattern ERG (PERG) and the photopic negative response (PhNR) of the ERG. For the PERG, longitudinal studies have been reported, suggesting that this method can be used for the early detection of glaucoma; for the PhNR, no longitudinal study is available as yet. The multifocal PERG can spatially resolve ganglion cell function but its glaucomatous reduction is typically panretinal, even with only local field changes and so, its topographic resolution is of no advantage in glaucoma. The multifocal visual evoked potential promises objective perimetry and shows sensitivity and specificity comparable with standard automated perimetry but has not been established as a routine tool to date.

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.

Fundus autofluorescence in carriers of choroideremia and correlation with electrophysiologic and psychophysical data.

PURPOSE: To describe fundus autofluorescence (FAF) in carriers of choroideremia (CHM), and to compare FAF findings with ophthalmoscopy and electrophysiologic and psychophysical data. DESIGN: Prospective, observational case series and systematic review. PARTICIPANTS: Six unrelated carriers of CHM. METHODS: Clinical examination included a comprehensive ophthalmic examination, fundus photography, FAF, kinetic perimetry, 2-color threshold perimetry (2CTP), full-field electroretinography (ERG), and multifocal ERG (mfERG). All 6 carriers (33-60 years of age) were screened for mutations in the coding region of Rab Escort Protein 1 gene (REP1) including close flanking intronic sequence and deletions within 2160 bp of 5' untranslated sequence. MAIN OUTCOME MEASURES: Intensity and distribution of FAF, rod sensitivity loss, cone sensitivity loss in 2CTP, amplitude and latency in full-field ERG, amplitude in mfERG, and correlation of all 3 parameters. RESULTS: Mutations in the coding region of REP1 were identified in 3 of 6 carriers. All 6 carriers had good visual acuity. Three carriers complained of photophobia and 1 of impaired vision in dim light. Ophthalmoscopy revealed peripapillary atrophy and retinal pigment epithelium (RPE) mottling mainly in the macular region, and additional RPE clumping and flecks of atrophy in the periphery. A very irregular pattern of low- and high-density FAF speckles was seen. Low-density FAF surrounding the optic nerve head corresponded with the peripapillary atrophy. In areas of major FAF changes, mfERG was deteriorated. The 2CTP images revealed functional disturbances in rods and cones. No general pattern was observed. On MfERG, reduced amplitudes in areas with normal cone sensitivity in 2CTP were seen. CONCLUSIONS: All 6 carriers of CHM showed a characteristic FAF pattern that can guide mutation analysis. Even when other functional testing is inconspicuous, FAF is a rapid, noninvasive indicator. FINANCIAL DISCLOSURE(S): The authors have no proprietary or commercial interest in any of the materials discussed in this article.

The mfERG response topography with scaled stimuli: effect of the stretch factor.

In multifocal electroretinogram recordings, the stimulus array is usually scaled with eccentricity to compensate for cone density changes. The strength of this scaling is parameterized by the so-called "stretch factor" (SF). In this study, we determined the quantitative influence of the SF striving for equal response densities over the entire stimulus area. VERIS 4.8 software was used to record multifocal ERGs from 11 normals with 61 hexagons and a 60 degrees stimulus diameter. Six recordings were obtained per subject with SFs 0, 12, 23, 25, 27, and 29. For analysis, we calculated the mean hexagon area per eccentricity and the average amplitude for elements of equal eccentricity (rings 1-5 = R1-R5), normalized to R2. As expected, recordings with SF = 0 showed the steepest amplitude drop-off with eccentricity (relative amplitudes of 1.90 (R1/R2), 1.0 (R2/R2), 0.67 (R3/R2), 0.51 (R4/R2), 0.38 (R5/R2). Using the preset SF = 12 in our set-up resulted in R1-amplitudes 1.7x larger than the peripheral amplitudes (relative amplitudes 1.39, 1.0, 0.88, 0.87, 0.82). SF = 23 compensated best for eccentricity, yielding relative amplitudes of 1.2, 1.0, 1.02, 1.08, and 1.11. Deviations increased again with higher SFs. To minimize amplitude variability over the stimulated retinal area, the optimal SF needs to be adjusted to the individual recording set-up.

Can we do without mydriasis in multifocal ERG recordings?

The purpose of this study is to determine whether mfERG recordings with natural pupils approximate recordings obtained in mydriasis if the stimulus luminance is adjusted. If so, a bright monitor could obviate pupil dilation, rendering clinical management considerably easier. In 11 eyes of six subjects, we took four mfERG recordings with a resolution of 61 scaled hexagons per eye, two with natural pupils and two in mydriasis with a luminance of 150 cd/m(2) and 500 cd/m(2), respectively. Conditions will be abbreviated as "LoNat, HiNat, LoDil, and HiDil," where Lo/Hi stands for low vs. high luminance and Nat/Dil stands for natural vs. dilated pupils. Response densities and peak times were averaged for the central seven and the remaining 54 hexagons. An ANOVA found highly significant effects for both amplitude and peak time between the four recording conditions (P always <0.001). However, for the two "middle" conditions, HiNat and LoDil, amplitudes and peak times did not differ markedly. Retinal illuminances of the conditions LoNat and HiNat each differed from the conditions LoDil and HiDil, respectively, by a factor of 5.4. Amplitudes increased and peak times decreased in the order of conditions LoNat, HiNat, LoDil, HiDil. When data were corrected for the Stiles-Crawford effect, there was an inversion of effective retinal illuminances of the recordings HiNat and LoDil. The mfERG guidelines correspond to the condition LoDil; for all practical purposes, the amplitudes and peak times are the same when the HiNat condition is used. Thus, when clinical constraints render pupil dilation undesirable, the mfERG can be recorded with natural pupils when monitor luminance is increased by five times (if available).

The fine structure of multifocal ERG topographies.

The multifocal electroretinogram (mfERG) allows for functional field mapping by concurrently deriving responses from a large number of retinal locations. The stimulus resolution most commonly used consists of 103 hexagonal elements. Here, we stimulated with an array of 509 elements. To determine the extent to which the multifocal ERG shows anatomical and physiological details, such as shadows cast by the retinal vasculature, we obtained mfERGs from two subjects using two different stimulus luminance levels and three light spectra. Good correspondence of some depressions with major blood vessels suggests relative angioscotomata. However, some reproducible local depressions cannot be attributed to blood vessel shadows cast on the retina, but more likely reflect local inhomogeneities in the physiological response characteristics.

Photopic negative response versus pattern electroretinogram in early glaucoma.

PURPOSE: Photopic negative response (PhNR) and pattern electroretinogram (PERG) are electrophysiological markers of retinal ganglion cell function; both are reduced in glaucoma. We compared PhNR and PERG in different stages of the disease. METHODS: Eleven eyes with preperimetric glaucoma (glaucomatous optic disc with normal field); 18 with manifest glaucoma; and 26 normals were included. We obtained PhNR (flash strength from 0.1-4 cd·s/m(2)) and steady-state PERG and analyzed PhNR amplitude (baseline to 72 ms trough); PhNR/b-wave ratio; PERG amplitude; and PERG ratio (0.8°/16°). RESULTS: Identification of PhNR structure was only reliable ≥1 cd·s/m(2) flash strength; amplitude and receiver operating characteristics (ROC) area under curve (AUC) changed little from 1 to 4 cd·s/m(2). Both PhNR and PERG (amplitude and ratio) were reduced in preperimetric and more so in manifest glaucoma. AUCs based on PhNR/PERG amplitudes were not significantly different from chance in preperimetric glaucoma (AUCs 0.61/0.59), but were significant in manifest glaucoma (0.78/0.76); ratios were significant in both glaucoma groups (0.80/0.73 and 0.80/0.79). In spite of that, PhNR ratio and PERG ratio were not significantly correlated (r = 0.22 across all groups); an ROC based on a combination of both reached AUCs of 0.85/0.90 for preperimetric/manifest glaucoma. CONCLUSIONS: Both PhNR and PERG performed similarly to detect glaucoma; for both, ratios performed better than amplitudes. The PhNR has the advantage of not requiring clear optics and refractive correction; the PERG has the advantage of being recorded with natural pupils.

Visualization of fundus vessel pulsation using principal component analysis.

PURPOSE: Spontaneous venous pulsation is one of the clinical signs with which to rule out elevated intracranial pressure and papilledema. More subtle pulsatile retinal movements are difficult to observe because of eye movements. Recording a fundus movie and aligning (registering) the images helps, but the images still contain distracting microsaccadic distortions and noise. The authors hypothesized that addressing these latter points should allow observation of minute pulsating features in fundus movies. METHODS: Principal component analysis (PCA), a basic form of blind source analysis, is applied to recorded fundus image sequences. The authors demonstrate this method in 5-second image sequences acquired with a near-infrared SLO (HRA+OCT Spectralis). The images are first registered to correct for eye drift, then microsaccade-distorted images are rejected, and the remaining image sequence is decomposed into principal components. Finally, a movie is constructed using the first five principal components (these had pulsatile features). RESULTS: Each of the processing steps (registration, cleaning, PCA filtering) improves the detection of pulsatile features, ultimately allowing clear visualization of spontaneous venous pulsation. Depending on the subject, additional features can be observed: pulsation amplitude of the arterial tree of approximately 10 µm, pulsation of arterioles down to 70-µm diameter, complete venous collapse, overall optic nerve head tissue pulsation, and mechanical links between veins and arteries. CONCLUSIONS: By disentangling pulsatile motion from other dynamic components of retinal images, unprecedented resolution in physiologic motion of retinal vessel structure is achievable.

ABCA4 and ROM1: implications for modification of the PRPH2-associated macular dystrophy phenotype.

PURPOSE: To identify the causative mutation leading to autosomal dominant macular dystrophy, cone dystrophy, and cone-rod dystrophy in a five-generation family and to explain the high intrafamilial phenotypic variation by identifying possible modifier genes. METHODS: Fifteen family members were investigated by detailed ophthalmic and electrophysiologic phenotyping. Mutation screening was initially performed with microarrays that detect known mutations in genes associated with retinal degeneration. Furthermore, the patients' genomic DNA was analyzed by sequencing analysis of PRPH2, ABCA4, and ROM1. RESULTS: Heterozygous mutations were identified in three genes and showed five different combinations within the studied family. All clearly affected family members carried the heterozygous PRPH2 mutation p.R172W. Patients with heterozygous sequence alterations only in ROM1 (p.R229H) or ABCA4 (p.V2050L) showed a mild ocular phenotype and were otherwise asymptomatic. The phenotypic severity of patients carrying the PRPH2 mutation increased with an additional mutation in ROM1. Patients carrying all three mutations were the most severely affected. CONCLUSIONS: Features of a PRPH2-associated phenotype might be modulated by additional mutations in other genes (in this family ABCA4 and/or ROM1) accounting for intrafamilial variability and resulting in a cumulative effect worsening the phenotype. Families showing a variable macular dystrophy phenotype caused by mutations in PRPH2 should be tested for additional mutations in ABCA4 and ROM1, as they may alter the progression of the PRPH2 phenotype. This testing will influence genetic counseling, as patients with additional mutations may be confronted with a faster progression of visual loss.

Genotyping microarray for CSNB-associated genes.

PURPOSE: Congenital stationary night blindness (CSNB) is a clinically and genetically heterogeneous retinal disease. Although electroretinographic (ERG) measurements can discriminate clinical subgroups, the identification of the underlying genetic defects has been complicated for CSNB because of genetic heterogeneity, the uncertainty about the mode of inheritance, and time-consuming and costly mutation scanning and direct sequencing approaches. METHODS: To overcome these challenges and to generate a time- and cost-efficient mutation screening tool, the authors developed a CSNB genotyping microarray with arrayed primer extension (APEX) technology. To cover as many mutations as possible, a comprehensive literature search was performed, and DNA samples from a cohort of patients with CSNB were first sequenced directly in known CSNB genes. Subsequently, oligonucleotides were designed representing 126 sequence variations in RHO, CABP4, CACNA1F, CACNA2D4, GNAT1, GRM6, NYX, PDE6B, and SAG and spotted on the chip. RESULTS: Direct sequencing of genes known to be associated with CSNB in the study cohort revealed 21 mutations (12 novel and 9 previously reported). The resultant microarray containing oligonucleotides, which allow to detect 126 known and novel mutations, was 100% effective in determining the expected sequence changes in all known samples assessed. In addition, investigation of 34 patients with CSNB who were previously not genotyped revealed sequence variants in 18%, of which 15% are thought to be disease-causing mutations. CONCLUSIONS: This relatively inexpensive first-pass genetic testing device for patients with a diagnosis of CSNB will improve molecular diagnostics and genetic counseling of patients and their families and gives the opportunity to analyze whether, for example, more progressive disorders such as cone or cone-rod dystrophies underlie the same gene defects.

Annular fundus autofluorescence abnormality in a case of macular dystrophy.

PURPOSE: To present a case of macular dystrophy with early changes in fundus autofluorescence. METHODS: A 20-year-old woman with a recent loss of visual acuity and onset of photophobia was examined. Color vision and visual field testing, fluorescein angiography, full-field and multifocal electroretinograms as well as fundus autofluorescence were performed. RESULTS: Best-corrected visual acuity was 20/100 (right eye) and 20/60 (left eye). There was a red-green color vision defect and a relative central scotoma in both eyes. Ophthalmoscopy and fluorescein angiography were essentially normal, the presence of a dark choroid was debatable. Full-field ERG responses were normal, but the multifocal ERG showed severely reduced responses in the macular region. Both eyes showed a slight circular parafoveolar increase of fundus autofluorescence. CONCLUSION: Besides multifocal ERG, fundus autofluorescence aids to objectively assess the manifestation of macular dystrophies but does not discern between different types in early stages.

Syndromic choroideremia: sublocalization of phenotypes associated with Martin-Probst deafness mental retardation syndrome.

PURPOSE: To identify the mutation leading to syndromic choroideremia (CHM) in two families and to define fundus autofluorescence (FAF) in CHM carriers. METHODS: The ophthalmic and clinical phenotype was investigated including FAF, neuropediatric, otorhinolaryngologic, cardiologic, and nephrologic examinations of three male patients (age, 11-46 years) and three female carriers (age, 11-46 years) from two families. Genomic DNA amplification (PCR) of the REP1 gene as well as adjacent loci was used to determine the molecular basis of the phenotype. RESULTS: Analysis of genomic DNA revealed large deletions that asymmetrically flank REP1 in both families, ranging from a minimum size of 6.3 and 8.5 mega base pairs (Mbp) to a maximum size of 9.7 and 14.1 Mbp, respectively. In addition to CHM, patients from these families exhibited mild syndromic features, including mental and motor retardation and low-frequency hearing loss. FAF showed a distinctive pattern characterized by small areas of reduced and increased autofluorescence in all female carriers. CONCLUSIONS: Both CHM families are the first to be described with large deletions that manifest with a mild syndromic phenotype. The location of the deletions indicates that they may allow sublocalization of the syndromic features to the most proximal region of X-linked distal spinal muscular atrophy (DSMAX) and Martin-Probst deafness mental retardation syndrome (MPDMRS). The FAF pattern is specific to CHM carriers and thus will help to identify and differentiate between carriers of other X-linked recessive carrier states such as in X-linked retinitis pigmentosa.

Disturbed visual system function in methionine synthase deficiency.

BACKGROUND: Isolated functional methionine synthase deficiency occurs in the cblE and cblG defects of methylcobalamin metabolism and is one of a number of causes of severely elevated plasma homocysteine. Clinical features are predominantly of a neurological nature but also include functional restriction of the visual system manifesting as loss of visual acuity and nystagmus. As yet, the origin and pathogenesis of impaired vision have not been explained. MATERIALS AND METHODS: We investigated a patient who was proven by complementation analysis in cultured fibroblasts to belong to the cblG complementation group. Ganzfeld electroretinograms (ERG) and flash visual evoked potentials (VEP) were recorded over a period of 4 years. RESULTS: Amplitudes of all International Society for Clinical Electrophysiology of Vision (ISCEV) standard responses were below normal. The greatest reductions were of rod response to 24 microV, of standard combined response (SC) b-wave to 120 microV, of oscillatory potentials (OP) to 5 microV, of cone response b-wave to 35 microV, and of 30 Hz flicker response to 8 microV. Except for SC and cone a-waves at age 2.5 and 3.5 years, as well as cone b-wave at 3.5 years, amplitudes remained at a subnormal level at follow-up examinations. Implicit times were slightly prolonged (SC b-wave 6 ms, OPs 2 ms, cone b-wave 2 ms, 30 Hz flicker 4 ms) or fell within the normal range. Responses of the flash VEP were severely deformed but reproducible. CONCLUSIONS: This is the first report of detailed investigations of the visual system in a patient with isolated methionine synthase deficiency. Reduced oscillatory potentials suggest microvascular damage to the retina through homocysteine. Decreased photoreceptor function as well as ganglion cell loss as indicated by pathological flash VEPs may reflect a cytotoxic impact of homocysteine on neurons of the visual pathway.

Heterozygous mutations of OTX2 cause severe ocular malformations.

Major malformations of the human eye, including microphthalmia and anophthalmia, are examples of phenotypes that recur in families yet often show no clear Mendelian inheritance pattern. Defining loci by mapping is therefore rarely feasible. Using a candidate-gene approach, we have identified heterozygous coding-region changes in the homeobox gene OTX2 in eight families with ocular malformations. The expression pattern of OTX2 in human embryos is consistent with the eye phenotypes observed in the patients, which range from bilateral anophthalmia to retinal defects resembling Leber congenital amaurosis and pigmentary retinopathy. Magnetic resonance imaging scans revealed defects of the optic nerve, optic chiasm, and, in some cases, brain. In two families, the mutations appear to have occurred de novo in severely affected offspring, and, in two other families, the mutations have been inherited from a gonosomal mosaic parent. Data from these four families support a simple model in which OTX2 heterozygous loss-of-function mutations cause ocular malformations. Four additional families display complex inheritance patterns, suggesting that OTX2 mutations alone may not lead to consistent phenotypes. The high incidence of mosaicism and the reduced penetrance have implications for genetic counseling.