Identifying haplotypes in recessive inherited retinal dystrophies using whole-genome linked-read sequencing.

Conventional next-generation sequencing methods, used in most gene panels, cannot separate maternally and paternally derived sequence information of distant variants. In recessive diseases, two or more equally plausible causative variants with unsolved phase information prevent accurate molecular diagnosis. In reality, close relatives might be unavailable for segregation analysis. Here, we utilized whole genome linked-read sequencing to assign variants to haplotypes in two patients with inherited retinal dystrophies. Patient 1 with macular dystrophy had variants c.3442T>C, p.(Cys1148Arg), c.4209G>T, p.(Glu1403Asp), and c.1182C>T, p.(Cys394=) in CRB1, and Patient 2 with nonsyndromic retinitis pigmentosa had c.1328T>A, p.(Val443Asp) and c.3032C>G, p.(Ser1011*) in AHI1. The relatives were not available for genotyping. Using whole genome linked-read sequencing we phased the variants to haplotypes providing genetic background for the retinal dystrophies. In future, when the price of sequencing methods that provides long-read data decreases and their read-depth and accuracy increases, they are probably considered the primary or adjunctive sequencing methods in genetic testing, allowing the immediate collection of phase information and thus obviating the need for the carrier testing and segregation analysis.

Photoreceptor cells as a source of fundus autofluorescence in recessive Stargardt disease.

Bisretinoid fluorophores form in photoreceptor outer segments from nonenzymatic reactions of vitamin A aldehyde. The short-wavelength autofluorescence (SW-AF) of fundus flecks in recessive Stargardt disease (STGD1) suggests a connection to these fluorophores. Through multimodal imaging, we sought to elucidate this link. Flecks observed in SW-AF images often colocalized with foci exhibiting reduced or absent near-infrared autofluorescence signal, the source of which is melanin in retinal pigment epithelial (RPE) cells. With serial imaging, changes in near-infrared autofluorescence (NIR-AF) preceded the onset of fleck hyperautofluorescence in SW-AF images and fleck profiles in NIR-AF images tended to be larger. Flecks in SW-AF and NIR-AF images also corresponded to hyperreflective lesions traversing photoreceptor-attributable bands in horizontal SD-OCT scans. The hyperreflective lesions interrupted adjacent OCT reflectivity bands and were associated with thinning of the outer nuclear layer. These SD-OCT findings are attributable to photoreceptor cell degeneration. Progressive increases and decreases in the SW-AF intensity of flecks were evident in color-coded quantitative fundus autofluorescence maps. In some cases, flecks appeared to spread radially from the fovea to approximately 8° of eccentricity, beyond which a circumferential spread characterized the distribution. Since the NIR-AF signal is derived from melanin and loss of this autofluorescence is indicative of RPE atrophy, the SW-AF of flecks cannot be accounted for by bisretinoid lipofuscin in RPE. Instead, we suggest that the bisretinoid serving as the source of the SW-AF signal, resides in photoreceptors, the cell that is also the site of bisretinoid synthesis.

Expression of class II cytokine genes in children's skin.

Immune regulation of the skin plays an important role in susceptibility and development of illnesses. The aim of our study was to localise the interleukin (IL)-10 family of cytokines, in children's skin and to determine possible age-related differences in the expression level. The mRNA expression level of IL10, IL19, IL20, IL22, IL24, IL26, IL28B, IL29 and their receptors IL10RA, IL10RB, IL20RA, IL20RB, IL22RA1, IL22RA2, IL28RA was compared in skin biopsies of children and adults and in childrens' skin cells by quantitative real-time PCR (qRT-PCR). Immunohistochemistry was performed to confirm the qRT-PCR findings. We found age-related differences in the expression of IL10RB, IL20, IL20RA, IL22RA1, IL22RA2, IL26 and IL28RA genes. Cell type-dependent expression of IL10 family cytokines was apparent in the skin. In addition to previously known differences in systemic immunological response of adults and children, the present results reveal differences in immune profile of adult and juvenile skin.

A retrospective longitudinal study of 52 Finnish patients with X-linked retinoschisis.

PURPOSE: To describe clinical characteristics in Finnish patients with X-linked retinoschisis (XLRS) longitudinally with emphasis on retinal morphology and genotype-phenotype correlations. METHODS: A retrospective cohort study reviewed medical records from patients with genetically confirmed XLRS from the Department of Ophthalmology, Helsinki University Hospital. Best-corrected visual acuity (BCVA), refraction, colour fundus photography, spectral-domain optical coherence tomography and genetic information were collected. RESULTS: Fifty-two males were diagnosed at the median age of 7 years (range 1-57) and followed for a median of 8 years (range, 1-49). Baseline findings included macular retinoschisis in 92 (89%), macular atrophy in 25 (24%) and peripheral retinoschisis in 22 (21%) eyes. Vitreous haemorrhage occurred in 10 (10%) eyes, more frequently with peripheral schisis (p < 0.001). Nearly half of the patients, 22 (42%) were classified as visually impaired according to WHO. Median central retinal thickness was similar between initial (355 µm) and latest visits (360 µm) (p = 0.781). Low BCVA was associated with macular atrophy (p < 0.001), ellipsoid zone disruption (p = 0.007) and peripheral retinoschisis (p = 0.006). The three Finnish founder mutations c.214G >A, c.221G >T, and c.325G >C in exon 4 of retinoschisin 1 (RS1) were identified in 40 patients (77%). No associations were found between the genotypes and phenotypes. CONCLUSION: Three-fourths of the patients carried the Finnish founder mutations in RS1, but we did not detect any genotype-phenotype association. Macular atrophy was associated with the poorest visual acuity. Ocular compilations were associated with peripheral retinoschisis, suggesting that these patients should be followed more frequently.

Insights Into PROM1-Macular Disease Using Multimodal Imaging.

Purpose: To describe the features of genetically confirmed PROM1-macular dystrophy in multimodal images. Methods: Thirty-six (36) eyes of 18 patients (5-66 years; mean age, 42.4 years) were prospectively studied by clinical examination and multimodal imaging. Short-wavelength autofluorescence (SW-AF) and quantitative fundus autofluorescence (qAF) images were acquired with a scanning laser ophthalmoscope (HRA+OCT, Heidelberg Engineering) modified by insertion of an internal autofluorescent reference. Further clinical testing included near-infrared autofluorescence (NIR-AF; HRA2, Heidelberg Engineering) with semiquantitative analysis, spectral domain-optical coherence tomography (HRA+OCT) and full-field electroretinography. All patients were genetically confirmed by exome sequencing. Results: All 18 patients presented with varying degrees of maculopathy. One family with individuals affected across two generations exhibited granular fleck-like deposits across the posterior pole. Areas of granular deposition in SW-AF and NIR-AF corresponded to intermittent loss of the ellipsoid zone, whereas discrete regions of hypoautofluorescence corresponded with a loss of outer retinal layers in spectral-domain optical coherence tomography scans. For 18 of the 20 eyes, qAF levels within the macula were within the 95% confidence intervals of healthy age-matched individuals; nor was the mean NIR-AF signal increased relative to healthy eyes. Conclusions: Although PROM1-macular dystrophy (Stargardt disease 4) can exhibit phenotypic overlap with recessive Stargardt disease, significantly increased SW-AF levels were not detected. As such, elevated bisretinoid lipofuscin may not be a feature of the pathophysiology of PROM1 disease. The qAF approach could serve as a method of early differential diagnosis and may help to identify appropriate disease targets as therapeutics become available to treat inherited retinal disease.

Lessons learned from quantitative fundus autofluorescence.

Quantitative fundus autofluorescence (qAF) is an approach that is built on a confocal scanning laser platform and used to measure the intensity of the inherent autofluorescence of retina elicited by short-wavelength (488 nm) excitation. Being non-invasive, qAF does not interrupt tissue architecture, thus allowing for structural correlations. The spectral features, cellular origin and topographic distribution of the natural autofluorescence of the fundus indicate that it is emitted from retinaldehyde-adducts that form in photoreceptor cells and accumulate, under most conditions, in retinal pigment epithelial cells. The distributions and intensities of fundus autofluorescence deviate from normal in many retinal disorders and it is widely recognized that these changing patterns can aid in the diagnosis and monitoring of retinal disease. The standardized protocol employed by qAF involves the normalization of fundus grey levels to a fluorescent reference installed in the imaging instrument. Together with corrections for magnification and anterior media absorption, this approach facilitates comparisons with serial images and images acquired within groups of patients. Here we provide a comprehensive summary of the principles and practice of qAF and we highlight recent efforts to elucidate retinal disease processes by combining qAF with multi-modal imaging.

Multimodal Imaging in Best Vitelliform Macular Dystrophy.

Purpose: In patients diagnosed with Best vitelliform macular dystrophy (BVMD), quantitative fundus autofluorescence (qAF), near-infrared fundus autofluorescence (NIR-AF), and spectral-domain optical coherence tomography (SD-OCT) were used to elucidate pathogenic mechanisms. Methods: Fourteen patients heterozygous for BEST1 mutations were recruited. qAF was analyzed using short-wavelength fundus autofluorescence (SW-AF) images. Mean gray levels (GL) were determined in nonlesion areas (7 to 9° eccentricity) and adjusted by GL measured in an internal fluorescent reference. NIR-AF images (787 nm; sensitivity of 96) were captured and saved in non-normalized mode. Horizontal SD-OCT images also were acquired and BVMD was staged according to the OCT findings. Results: In the pre-vitelliform stage, NIR-AF imaging revealed an area of reduced fluorescence, whereas in the vitelliruptive stage, puncta of elevated NIR-AF signal were present. In both SW-AF and NIR-AF images, the vitelliform lesion in the atrophic stage was marked by reduced signal. At all stages of BVMD, nonlesion qAF was within the 95% confidence intervals for healthy eyes. Similarly, the NIR-AF intensity measurements outside the vitelliform lesion were comparable to the healthy control eye. SD-OCT scans revealed a fluid-filled detachment between the ellipsoid zone and the hyperreflectivity band attributable to RPE/Bruch's membrane. Conclusions: NIR-AF imaging can identify the pre-vitelliform stage of BVMD. Mutations in BEST1 are not associated with increased levels of SW-AF outside the vitelliform lesion. Elevated SW-AF within the fluid-filled lesion likely reflects the inability of RPE to phagocytose outer segments due to separation of RPE from photoreceptor cells, together with progressive photoreceptor cell impairment.

Patterns and Intensities of Near-Infrared and Short-Wavelength Fundus Autofluorescence in Choroideremia Probands and Carriers.

Purpose: To ascertain cellular constituents within islands of preserved retina in choroideremia (CHM) by multimodal imaging. Methods: CHM probands (16) and female carriers (9) of CHM were studied. Near-infrared autofluorescence (NIR-AF; 787-nm excitation; emission, >830 nm), short-wavelength autofluorescence (SW-AF; 488-nm excitation, 500- to 680-nm emission), and spectral-domain optical coherence tomography (SD-OCT) images were acquired with a confocal scanning laser ophthalmoscope. SW-AF intensities were measured by quantitative fundus autofluorescence (qAF), and NIR-AF intensity profiles were analyzed. Retinal thicknesses and visual acuity were measured. Results: In 19 of 31 eyes of affected males, islands of preserved NIR-AF signal were also visible as fluorescence signal in SW-AF images. Notable in 12 eyes were areas of speckled SW-AF that was hypoautofluorescent in the NIR-AF image. Islands of preserved NIR-AF and SW-AF signal were often associated with the presence of visible but thinned outer nuclear layer and discontinuous interdigitation zone, ellipsoid zone, and external limiting membrane. NIR-AF profiles revealed that even in areas of preserved retina, the NIR-AF signal from retinal pigment epithelium (RPE) melanin is greatly reduced. qAF was reduced overall. The fundus of carriers was characterized by a mosaicism in which patches of reduced NIR-AF colocalized with reduced SW-AF. Conclusions: In CHM-affected males, the presence of RPE was indicated by an NIR-AF signal and the absence of hypertransmission of OCT signal into the choroid. RPE preservation was associated with better visual acuity. In carriers, patches of reduced SW-AF colocalized with decreased NIR-AF and qAF was severely reduced.

Hyperautofluorescent Dots are Characteristic in Ceramide Kinase Like-associated Retinal Degeneration.

There is a lack of studies which seek to discern disease expression in patients with mutations that alter retinal ceramide metabolism, specifically in the ceramide kinase like (CERKL) gene. This cross-sectional case series reports a novel phenotypic manifestation of CERKL-associated retinopathy. Four unrelated patients with homozygous CERKL mutations underwent a complete ocular exam, spectral-domain optical coherence tomography, short-wavelength fundus autofluorescence (SW-AF), quantitative autofluorescence (qAF), and full-field electroretinogram (ffERG). Decreased visual acuity and early-onset maculopathy were present in all patients. All four patients had extensive hyperautofluorescent foci surrounding an area of central atrophy on SW-AF imaging, which has not been previously characterized. An abnormal spatial distribution of qAF signal was seen in one patient, and abnormally elevated qAF8 signal in another patient. FfERG recordings showed markedly attenuated rod and cone response in all patients. We conclude that these patients exhibit several features that, collectively, may warrant screening of CERKL as a first candidate: early-onset maculopathy, severe generalized retinal dysfunction, peripheral lacunae, intraretinal pigment migration, and hyperautofluorescent foci on SW-AF.

Spectrum of Disease Severity and Phenotype in Choroideremia Carriers.

PURPOSE: To characterize and bring awareness to the disease spectrum of female choroideremia patients, as severity can vary from mild to severe disease, comparable to that observed in male patients. DESIGN: Retrospective cohort study. METHODS: Twelve female carriers of disease-causing variants in the CHM gene confirmed by molecular genetic sequencing were characterized clinically and imaged with short-wave fundus autofluorescence (SW-FAF), spectral-domain optical coherence tomography (OCT), and color fundus imaging. RESULTS: Twelve unrelated female patients with a clinical and genetic diagnosis of choroideremia carriers were included in this study. Disease severity among these phenotypes ranged from mild to severe, resembling the typical presentation of choroideremia in male patients. Mild disease presented with retinal pigment epithelium mottling, a patchy pattern of hypoautofluorescent speckles on SW-FAF, and intact retinal layers on spectral-domain OCT. Severe disease presented with widespread chorioretinal atrophy as shown by SW-FAF and spectral-domain OCT. Each of the identified genetic variants in CHM was predicted to be disease-causing according to in silico prediction software. Disease progression analysis of 4 patients with follow-up showed a decline in visual acuity for 2 patients, with progression observed on spectral-domain OCT in 1 of the patients. No significant disease progression on SW-FAF was observed for any of the patients. CONCLUSIONS: Female carriers of choroideremia can present with a wide range of clinical phenotypes and disease severity, from mild to severe disease, similar to male subjects. Symptomatic female subjects should be considered for current and upcoming gene replacement therapy clinical trials.

Modification of the PROM1 disease phenotype by a mutation in ABCA4.

Background: The extensive phenotypic heterogeneity of monogenic diseases can be largely traced to intragenic variation; however, recent advances in clinical detection and gene sequencing have uncovered the emerging role of non-allelic variation (i.e. genetic trans-modifiers) in shaping disease phenotypes. Identifying these associations are not only of significant diagnostic value, but also provides scientific insight into the expanded molecular etiology of rare diseases. This reports describes the discordant clinical manifestation of a family segregating mutations in ABCA4 and PROM1. Methods: Three patients across a two generation family underwent multimodal imaging and functional testing of the retina including color photography, fundus autofluorescence (AF), spectral domain-optical coherence tomography (SD-OCT) and full-field electroretinography (ffERG). Genetic characterization was carried out by direct Sanger and whole exome sequencing. Results: Clinical examination revealed similar retinal degenerative phenotypes in the proband and her mother. Despite being younger, the proband's phenotype was more advanced and exhibited additional features related to Stargardt disease not found in the mother. Whole exome sequencing identified a pathogenic missense variant in PROM1, c.400C > T, p.(Arg134Cys), as the underlying cause of retinal disease in both the proband and mother. Sequencing of the ABCA4 locus uncovered a single disease-causing variant, c.5714 + 5G > A in the daughter segregating from the father who, surprisingly, also exhibited very subtle disease changes associated with STGD1 despite being a heterozygous carrier. Conclusions: Harboring an additional heterozygous ABCA4 mutation increases severity and confers STGD1-like features in patients with PROM1 disease which provides supporting evidence for their shared pathophysiology and potential treatment prospects.

Mutations in GPR143/OA1 and ABCA4 Inform Interpretations of Short-Wavelength and Near-Infrared Fundus Autofluorescence.

Purpose: We sought to advance interpretations and quantification of short-wavelength fundus autofluorescence (SW-AF) emitted from bisretinoid lipofuscin and near-infrared autofluoresence (NIR-AF) originating from melanin. Methods: Carriers of mutations in X-linked GPR143/OA1, a common form of ocular albinism; patients with confirmed mutations in ABCA4 conferring increased SW-AF; and subjects with healthy eyes were studied. SW-AF (488 nm excitation, 500-680 nm emission) and NIR-AF (excitation 787 nm, emission >830 nm) images were acquired with a confocal scanning laser ophthalmoscope. SW-AF images were analyzed for quantitative autofluoresence (qAF). Analogous methods of image acquisition and analysis were performed in albino and pigmented Abca4-/- mice and wild-type mice. Results: Quantitation of SW-AF (qAF), construction of qAF color-coded maps, and examination of NIR-AF images from GPR143/OA1 carriers revealed mosaics in which patches of fundus exhibiting NIR-AF signal had qAF levels within normal limits whereas the hypopigmented areas in the NIR-AF image corresponded to foci of elevated qAF. qAF also was increased in albino versus pigmented mice. Although melanin contributes to fundus infrared reflectance, the latter appeared to be uniform in en face reflectance images of GPR143/OA1-carriers. In patients diagnosed with ABCA4-associated disease, NIR-AF increased in tandem with increased qAF originating in bisretinoid lipofuscin. Similarly in Abca4-/- mice having increased SW-AF, NIR-AF was more pronounced than in wild-type mice. Conclusions: These studies corroborate RPE melanin as the major source of NIR-AF but also indicate that bisretinoid lipofuscin, when present at sufficient concentrations, contributes to the NIR-AF signal. Ocular melanin attenuates the SW-AF signal.

Intraretinal Correlates of Reticular Pseudodrusen Revealed by Autofluorescence and En Face OCT.

Purpose: We sought to determine whether information revealed from the reflectance, autofluorescence, and absorption properties of RPE cells situated posterior to reticular pseudodrusen (RPD) could provide insight into the origins and structure of RPD. Methods: RPD were studied qualitatively by near-infrared fundus autofluorescence (NIR-AF), short-wavelength fundus autofluorescence (SW-AF), and infrared reflectance (IR-R) images, and the presentation was compared to horizontal and en face spectral domain optical coherence tomographic (SD-OCT) images. Images were acquired from 23 patients (39 eyes) diagnosed with RPD (mean age 80.7 ± 7.1 [SD]; 16 female; 4 Hispanics, 19 non-Hispanic whites). Results: In SW-AF, NIR-AF, and IR-R images, fundus RPD were recognized as interlacing networks of small scale variations in IR-R and fluorescence (SW-AF, NIR-AF) intensities. Darkened foci of RPD colocalized in SW-AF and NIR-AF images, and in SD-OCT images corresponded to disturbances of the interdigitation (IZ) and ellipsoid (EZ) zones and to more pronounced hyperreflective lesions traversing photoreceptor-attributable bands in SD-OCT images. Qualitative assessment of the outer nuclear layer (ONL) revealed thinning as RPD extended radially from the outer to inner retina. In en face OCT, hyperreflective areas in the EZ band correlated topographically with hyporeflective foci at the level of the RPE. Conclusions: The hyperreflective lesions corresponding to RPD in SD-OCT scans are likely indicative of degenerating photoreceptor cells. The darkened foci at positions of RPD in NIR-AF and en face OCT images indicate changes in the RPE monolayer with the reduced NIR-AF and en face OCT signal suggesting a reduction in melanin that could be accounted for by RPE thinning.