PRPH2-Related Retinal Dystrophies: Mutational Spectrum in 103 Families from a Spanish Cohort.

PRPH2, one of the most frequently inherited retinal dystrophy (IRD)-causing genes, implies a high phenotypic variability. This study aims to analyze the PRPH2 mutational spectrum in one of the largest cohorts worldwide, and to describe novel pathogenic variants and genotype-phenotype correlations. A study of 220 patients from 103 families recruited from a database of 5000 families. A molecular diagnosis was performed using classical molecular approaches and next-generation sequencing. Common haplotypes were ascertained by analyzing single-nucleotide polymorphisms. We identified 56 variants, including 11 novel variants. Most of them were missense variants (64%) and were located in the D2-loop protein domain (77%). The most frequently occurring variants were p.Gly167Ser, p.Gly208Asp and p.Pro221_Cys222del. Haplotype analysis revealed a shared region in families carrying p.Leu41Pro or p.Pro221_Cys222del. Patients with retinitis pigmentosa presented an earlier disease onset. We describe the largest cohort of IRD families associated with PRPH2 from a single center. Most variants were located in the D2-loop domain, highlighting its importance in interacting with other proteins. Our work suggests a likely founder effect for the variants p.Leu41Pro and p.Pro221_Cys222del in our Spanish cohort. Phenotypes with a primary rod alteration presented more severe affectation. Finally, the high phenotypic variability in PRPH2 hinders the possibility of drawing genotype-phenotype correlations.

KCNV2-associated retinopathy: genotype-phenotype correlations - KCNV2 study group report 3.

BACKGROUND/AIMS: To investigate genotype-phenotype associations in patients with KCNV2 retinopathy. METHODS: Review of clinical notes, best-corrected visual acuity (BCVA), molecular variants, electroretinography (ERG) and retinal imaging. Subjects were grouped according to the combination of KCNV2 variants-two loss-of-function (TLOF), two missense (TM) or one of each (MLOF)-and parameters were compared. RESULTS: Ninety-two patients were included. The mean age of onset (mean±SD) in TLOF (n=55), TM (n=23) and MLOF (n=14) groups was 3.51±0.58, 4.07±2.76 and 5.54±3.38 years, respectively. The mean LogMAR BCVA (±SD) at baseline in TLOF, TM and MLOF groups was 0.89±0.25, 0.67±0.38 and 0.81±0.35 for right, and 0.88±0.26, 0.69±0.33 and 0.78±0.33 for left eyes, respectively. The difference in BCVA between groups at baseline was significant in right (p=0.03) and left eyes (p=0.035). Mean outer nuclear layer thickness (±SD) at baseline in TLOF, MLOF and TM groups was 37.07±15.20 µm, 40.67±12.53 and 40.38±18.67, respectively, which was not significantly different (p=0.85). The mean ellipsoid zone width (EZW) loss (±SD) was 2051 µm (±1318) for patients in the TLOF, and 1314 µm (±965) for MLOF. Only one patient in the TM group had EZW loss at presentation. There was considerable overlap in ERG findings, although the largest DA 10 ERG b-waves were associated with TLOF and the smallest with TM variants. CONCLUSIONS: Patients with missense alterations had better BCVA and greater structural integrity. This is important for patient prognostication and counselling, as well as stratification for future gene therapy trials.

Comprehensive Genotyping and Phenotyping Analysis of GUCY2D-Associated Rod- and Cone-Dominated Dystrophies.

PURPOSE: To describe the genetic and clinical spectrum of GUCY2D-associated retinopathies and to accurately establish their prevalence in a large cohort of patients. DESIGN: Retrospective case series. METHODS: Institutional study of 47 patients from 27 unrelated families with retinal dystrophies carrying disease-causing GUCY2D variants from the Fundación Jiménez Díaz hospital dataset of 8000 patients. Patients underwent ophthalmological examination and molecular testing by Sanger or exome sequencing approaches. Statistical and principal component analyses were performed to determine genotype-phenotype correlations. RESULTS: Four clinically different associated phenotypes were identified: 66.7% of families with cone/cone-rod dystrophy, 22.2% with Leber congenital amaurosis, 7.4% with early-onset retinitis pigmentosa, and 3.7% with congenital night blindness. Twenty-three disease-causing GUCY2D variants were identified, including 6 novel variants. Biallelic variants accounted for 28% of patients, whereas most carried dominant alleles associated with cone/cone-rod dystrophy. The disease onset had statistically significant differences according to the functional variant effect. Patients carrying GUCY2D variants were projected into 3 subgroups by allelic combination, disease onset, and presence of nystagmus or night blindness. In contrast to patients with the most severe phenotype of Leber congenital amaurosis, 7 patients with biallelic GUCY2D had a later and milder rod form with night blindness in infancy as the first symptom. CONCLUSIONS: This study represents the largest GUCY2D cohort in which 4 distinctly different phenotypes were identified, including rare intermediate presentations of rod-dominated retinopathies. We established that GUCY2D is linked to about 1% of approximately 3000 molecularly characterized families of our cohort. All of these findings are critical for defining cohorts for inclusion in future clinical trials.

Application of multicolour reflectance imaging for the characterisation of inherited retinal disorders.

AIM: To describe the role of multicolour reflectance images (MCI) in the phenotypic diagnosis of inherited retinal disorders (IRDs). METHODS: A retrospective review of consecutive patients affected by IRDs examined with MCI techniques from January to December 2019 at a tertiary care referral centre. All patients had MCI, fundus autofluorescence and optical coherence tomography taken at the same time point. The ability of each modality to highlight clinical features was assessed. Lesions' size was also measured and compared among imaging modalities. RESULTS: Thirty eyes of 15 patients were included in the study, 6 males and 9 females, with a mean age of 44 years (range: 19-57.5). The most frequent clinical diagnosis were: pattern dystrophies, and late-onset retinal degeneration. Next-generation or Sanger sequencing analysis was carried out in all patients. Blue and green reflectance were relevant in highlighting peripheral mottling in fundus albipunctatus, pseudoreticular drusen in late-onset retinal degeneration, parafoveal hyperreflective area in bull's eye maculopathy and crystals in Bietti's crystalline dystrophy. Likewise, it is to mention the ability of infrared reflectance to detect hyperreflective patches in posterior pole in neurofibromatosis type 1 and retinal changes in pattern dystrophies and cone dystrophies. CONCLUSION: Multicolour imaging technique enables the detection of clinical features that could be overlooked by other imaging modalities, allowing accurate phenotypic characterisation of IRDs and guiding genetic diagnose, and may become a meaningful monitoring tool for future treatments.

Allelic overload and its clinical modifier effect in Bardet-Biedl syndrome.

Bardet-Biedl syndrome (BBS) is an autosomal recessive ciliopathy characterized by extensive inter- and intra-familial variability, in which oligogenic interactions have been also reported. Our main goal is to elucidate the role of mutational load in the clinical variability of BBS. A cohort of 99 patients from 77 different families with biallelic pathogenic variants in a BBS-associated gene was retrospectively recruited. Human Phenotype Ontology terms were used in the annotation of clinical symptoms. The mutational load in 39 BBS-related genes was studied in index cases using different molecular and next-generation sequencing (NGS) approaches. Candidate allele combinations were analysed using the in silico tools ORVAL and DiGePred. After clinical annotation, 76 out of the 99 cases a priori fulfilled established criteria for diagnosis of BBS or BBS-like. BBS1 alleles, found in 42% of families, were the most represented in our cohort. An increased mutational load was excluded in 41% of the index cases (22/54). Oligogenic inheritance was suspected in 52% of the screened families (23/45), being 40 tested by means of NGS data and 5 only by traditional methods. Together, ORVAL and DiGePred platforms predicted an oligogenic effect in 44% of the triallelic families (10/23). Intrafamilial variable severity could be clinically confirmed in six of the families. Our findings show that the presence of more than two alleles in BBS-associated genes correlated in six families with a more severe phenotype and associated with specific findings, highlighting the role of the mutational load in the management of BBS cases.

Impact of Next Generation Sequencing in Unraveling the Genetics of 1036 Spanish Families With Inherited Macular Dystrophies.

Purpose: To assess the potential of next-generation sequencing (NGS) technologies to characterize cases diagnosed with autosomal recessive (ar) or sporadic (s) macular dystrophies (ar/sMD) and describe their mutational spectrum. Methods: A cohort of 1036 families was classified according to their suspected clinical diagnosis-Stargardt disease (STGD), cone and cone-rod dystrophy (CCRD) or other maculopathies (otherMD). Molecular studies included genotyping microarrays, Sanger sequencing, NGS, and sequencing of intronic regions of the ABCA4 gene. Clinical reclassification was done after the genetic study. Results: At the end of the study, 677 patients (65%) had a confirmed genetic diagnosis, representing 78%, 63%, and 38% of STGD, CCRD, and otherMD groups of patients, respectively. ABCA4 is the most mutated gene in all groups, and a second pathogenic variant was found in 76% of STGD patients with one previously identified mutated ABCA4 allele. Autosomal dominant or X-linked mutations were found in 5% of cases together with not-MD genes (CHM, EYS, RHO, RPGR, RLBP1, OPA1, and USH2A among others) leading to their reclassification. Novel variants in the very rare genes PLA2G5 and TTLL5 revealed additional phenotypic associations. Conclusions: This study provides for the first time a genetic landscape of 1036 ar/sMD families according to their suspected diagnosis. The analysis of >200 genes associated with retinal dystrophies and the entire locus of ABCA4 increase the rate of characterization, even regardless of available clinical and familiar data. The use of the suspected a priori diagnosis referred by the clinicians, especially in the past, could lead to clinical reclassifications to other inherited retinal dystrophies.

RPE65-related retinal dystrophy: Mutational and phenotypic spectrum in 45 affected patients.

INTRODUCTION: Biallelic pathogenic RPE65 variants are related to a spectrum of clinically overlapping inherited retinal dystrophies (IRD). Most affected individuals progress to severe disease, with 50% of patients becoming legally blind by 20 years of age. Deeper knowledge of the mutational spectrum and the phenotype-genotype correlation in RPE65-related IRD is needed. PATIENTS AND METHODS: Forty-five affected subjects from 27 unrelated families with a clinical diagnosis of RPE65-related IRD were included. Clinical evaluation consisted of self-reported ophthalmological history and objective ophthalmological examination. Patients' genotype was classified according to variant class (truncating or missense) or to variant location at different protein domains. The main phenotypic outcome measure was age at onset (AAO) of symptomatic disease and a Kaplan-Meier analysis of disease symptom event-free survival was performed. RESULTS: Twenty-nine different RPE65 variants were identified in our cohort, 7 of them novel. Patients carrying two missense alleles showed a later disease onset than those with 1 or 2 truncating variants (log-rank test p <0.05). While 60% of patients carrying a missense/missense genotype presented symptoms before or during the first year of life, almost all patients with at least 1 truncating allele (91%) had an AAO ≤1 year (p <0.05). CONCLUSION: Our findings suggest an association between the type of RPE65 variant carried and AAO. These findings provide useful data on RPE65-associated IRD phenotypes and may help improve clinical and therapeutic management of these patients.

KCNV2-Associated Retinopathy: Detailed Retinal Phenotype and Structural Endpoints-KCNV2 Study Group Report 2.

PURPOSE: To describe the detailed retinal phenotype of KCNV2-associated retinopathy. STUDY DESIGN: Multicenter international retrospective case series. METHODS: Review of retinal imaging including fundus autofluorescence (FAF) and optical coherence tomography (OCT), including qualitative and quantitative analyses. RESULTS: Three distinct macular FAF features were identified: (1) centrally increased signal (n = 35, 41.7%), (2) decreased autofluorescence (n = 27, 31.1%), and (3) ring of increased signal (n = 37, 44.0%). Five distinct FAF groups were identified based on combinations of those features, with 23.5% of patients changing the FAF group over a mean (range) follow-up of 5.9 years (1.9-13.1 years). Qualitative assessment was performed by grading OCT into 5 grades: (1) continuous ellipsoid zone (EZ) (20.5%); (2) EZ disruption (26.1%); (3) EZ absence, without optical gap and with preserved retinal pigment epithelium complex (21.6%); (4) loss of EZ and a hyporeflective zone at the foveola (6.8%); and (5) outer retina and retinal pigment epithelium complex loss (25.0%). Eighty-six patients had scans available from both eyes, with 83 (96.5%) having the same grade in both eyes, and 36.1% changed OCT grade over a mean follow-up of 5.5 years. The annual rate of outer nuclear layer thickness change was similar for right and left eyes. CONCLUSIONS: KCNV2-associated retinopathy is a slowly progressive disease with early retinal changes, which are predominantly symmetric between eyes. The identification of a single OCT or FAF measurement as an endpoint to determine progression that applies to all patients may be challenging, although outer nuclear layer thickness is a potential biomarker. Findings suggest a potential window for intervention until 40 years of age.

Genotype-phenotype correlation in patients with Usher syndrome and pathogenic variants in MYO7A: implications for future clinical trials.

PURPOSE: We aimed to establish correlations between the clinical features of a cohort of Usher syndrome (USH) patients with pathogenic variants in MYO7A, type of pathogenic variant, and location on the protein domain. METHODS: Sixty-two USH patients from 46 families with biallelic variants in MYO7A were examined for visual and audiological features. Participants were evaluated based on self-reported ophthalmological history and ophthalmological investigations (computerized visual field testing, best-corrected visual acuity, and ophthalmoscopic and electrophysiological examination). Optical coherence tomography and fundus autofluorescence imaging were performed when possible. Auditory and vestibular functions were evaluated. Patients were classified according to the type of variant and the protein domain where the variants were located. RESULTS: Most patients displayed a typical USH1 phenotype, that is, prelingual severe-profound sensorineural hearing loss, prepubertal retinitis pigmentosa (RP) and vestibular dysfunction. No statistically significant differences were observed for the variables analysed except for the onset of hearing loss due to the existence of two USH2 cases, defined as postlingual sensorineural hearing loss, postpubertal onset of RP, and absence of vestibular dysfunction, and one atypical case of USH. CONCLUSION: We were unable to find a correlation between genotype and phenotype for MYO7A. However, our findings could prove useful for the assessment of efficacy in clinical trials, since the type of MYO7A variant does not seem to change the onset, severity or course of visual disease.

Multicolor Imaging for the Detection of Inner Nuclear Layer Microcysts Secondary to Optic Nerve Atrophy.

ABSTRACT: Inner nuclear layer (INL) microcysts have been reported in diseases affecting the optic nerve. The new ocular imaging techniques detect minimal structural alterations at the macula and correlate these findings to different etiologies with less invasive procedures. The relationship between ganglion cells distribution at the macula and chiasmal nerve fibers enables the diagnosis and location of neurological lesions by new generation optical coherence tomography (SD-OCT) imaging devices. We report the evaluation of a patient with a history of optic nerve trauma and macular INL microcysts with multicolor SD-OCT technology that shows a pattern that localizes the lesion to the left optic nerve and proximal segment of the chiasm.

Genetic landscape of 6089 inherited retinal dystrophies affected cases in Spain and their therapeutic and extended epidemiological implications.

Inherited retinal diseases (IRDs), defined by dysfunction or progressive loss of photoreceptors, are disorders characterized by elevated heterogeneity, both at the clinical and genetic levels. Our main goal was to address the genetic landscape of IRD in the largest cohort of Spanish patients reported to date. A retrospective hospital-based cross-sectional study was carried out on 6089 IRD affected individuals (from 4403 unrelated families), referred for genetic testing from all the Spanish autonomous communities. Clinical, demographic and familiar data were collected from each patient, including family pedigree, age of appearance of visual symptoms, presence of any systemic findings and geographical origin. Genetic studies were performed to the 3951 families with available DNA using different molecular techniques. Overall, 53.2% (2100/3951) of the studied families were genetically characterized, and 1549 different likely causative variants in 142 genes were identified. The most common phenotype encountered is retinitis pigmentosa (RP) (55.6% of families, 2447/4403). The most recurrently mutated genes were PRPH2, ABCA4 and RS1 in autosomal dominant (AD), autosomal recessive (AR) and X-linked (XL) NON-RP cases, respectively; RHO, USH2A and RPGR in AD, AR and XL for non-syndromic RP; and USH2A and MYO7A in syndromic IRD. Pathogenic variants c.3386G > T (p.Arg1129Leu) in ABCA4 and c.2276G > T (p.Cys759Phe) in USH2A were the most frequent variants identified. Our study provides the general landscape for IRD in Spain, reporting the largest cohort ever presented. Our results have important implications for genetic diagnosis, counselling and new therapeutic strategies to both the Spanish population and other related populations.

KCNV2-Associated Retinopathy: Genetics, Electrophysiology, and Clinical Course-KCNV2 Study Group Report 1.

PURPOSE: To investigate genetics, electrophysiology, and clinical course of KCNV2-associated retinopathy in a cohort of children and adults. STUDY DESIGN: This was a multicenter international clinical cohort study. METHODS: Review of clinical notes and molecular genetic testing. Full-field electroretinography (ERG) recordings, incorporating the international standards, were reviewed and quantified and compared with age and recordings from control subjects. RESULTS: In total, 230 disease-associated alleles were identified from 117 patients, corresponding to 75 different KCNV2 variants, with 28 being novel. The mean age of onset was 3.9 years old. All patients were symptomatic before 12 years of age (range, 0-11 years). Decreased visual acuity was present in all patients, and 4 other symptoms were common: reduced color vision (78.6%), photophobia (53.5%), nyctalopia (43.6%), and nystagmus (38.6%). After a mean follow-up of 8.4 years, the mean best-corrected visual acuity (BCVA ± SD) decreased from 0.81 ± 0.27 to 0.90 ± 0.31 logarithm of minimal angle of resolution. Full-field ERGs showed pathognomonic waveform features. Quantitative assessment revealed a wide range of ERG amplitudes and peak times, with a mean rate of age-associated reduction indistinguishable from the control group. Mean amplitude reductions for the dark-adapted 0.01 ERG, dark-adapted 10 ERG a-wave, and LA 3.0 30 Hz and LA3 ERG b-waves were 55%, 21%, 48%, and 74%, respectively compared with control values. Peak times showed stability across 6 decades. CONCLUSION: In KCNV2-associated retinopathy, full-field ERGs are diagnostic and consistent with largely stable peripheral retinal dysfunction. Report 1 highlights the severity of the clinical phenotype and established a large cohort of patients, emphasizing the unmet need for trials of novel therapeutics.

Genotype-Phenotype Correlations in a Spanish Cohort of 506 Families With Biallelic ABCA4 Pathogenic Variants.

PURPOSE: To define genotype-phenotype correlations in the largest cohort study worldwide of patients with biallelic ABCA4 variants, including 434 patients with Stargardt disease (STGD1) and 72 with cone-rod dystrophy (CRD). DESIGN: Cohort study. METHODS: We characterized 506 patients with ABCA4 variants using conventional genetic tools and next-generation sequencing technologies. Medical history and ophthalmologic data were obtained from 372 patients. Genotype-phenotype correlation studies were carried out for the following variables: variant type, age at symptom onset (AO), and clinical phenotype. RESULTS: A total of 228 different pathogenic variants were identified in 506 ABCA4 patients, 50 of which were novel. Genotype-phenotype correlations showed that most of the patients with biallelic truncating variants presented with CRD and that these cases had a significantly earlier AO than patients with STGD1. Three missense variants are associated with CRD for the first time (c.1804C>T; p.[Arg602Trp], c.3056C>T; p.[Thr1019Met], and c.6320G>C; p.[Arg2107Pro]). Analysis of the most prevalent ABCA4 variant in Spain, c.3386G>T; p.(Arg1129Leu), revealed that is correlated to STGD1, later AO, and foveal sparing. CONCLUSIONS: Our study, conducted in the largest ABCA4-associated disease cohort reported to date, updates the genotype-phenotype model established for ABCA4 variants and broadens the mutational spectrum of the gene. According to our observations, patients with ABCA4 presenting with 2 truncating variants may first present features of STGD1 but eventually develop rod dysfunction, and specific missense variants may be associated with a different phenotype, underscoring the importance of an accurate genetic diagnosis. Also, it is a prerequisite for enrollment in clinical trials, and to date, no other treatment has been approved for STGD1.

Expanded Phenotypic Spectrum of Retinopathies Associated with Autosomal Recessive and Dominant Mutations in PROM1.

PURPOSE: To describe the genetic and phenotypic characteristics of a cohort of patients with PROM1 variants. DESIGN: Case-case study. METHODS: We screened a cohort of 2216 families with inherited retinal dystrophies using classical molecular techniques and next-generation sequencing approaches. The clinical histories of 25 patients were reviewed to determine age of onset of symptoms and the results of ophthalmoscopy, best-corrected visual acuity, full-field electroretinography, and visual field studies. Fundus autofluorescence and spectral-domain optical coherence tomography were further assessed in 7 patients. RESULTS: PROM1 variants were identified in 32 families. Disease-causing variants were found in 18 autosomal recessive and 4 autosomal dominant families. Monoallelic pathogenic variants or variants of unknown significance were identified in the remaining 10 families. Comprehensive phenotyping of 25 patients from 22 families carrying likely disease-causing variants revealed clinical heterogeneity associated with the PROM1 gene. Most of these patients presented cone-rod dystrophy and some exhibited macular dystrophy or retinitis pigmentosa, while all presented with macular damage. Phenotypic association of a dominant splicing variant with late-onset mild maculopathy was established. This variant is one of the 3 likely founder variants identified in our Spanish cohort. CONCLUSIONS: We report the largest cohort of patients with PROM1 variants, describing in detail the phenotype in 25 of them. Interestingly, within the variability of phenotypes related to this gene, macular involvement is a common feature in all patients.

A large study reveals no association between APOE and Parkinson's disease.

BACKGROUND: Research focusing on the role of APOE in Parkinson's disease (PD) has been largely inconclusive, creating a broad discrepancy in association studies. OBJECTIVE: To elucidate the role of APOE alleles in PD risk by studying a large sample size and controlling for population substructure. PATIENTS AND METHODS: In total, 3465 case and control samples were genotyped, obtained from the NINDS Neurogenetics repository. RESULTS: No significant differences in ε4 dosages exist between PD cases and controls. The frequency of ε4 carriers differed slightly between cases and controls at 24% (580/2412) and 26% (270/1053), respectively. Likewise, mean dosages of APOE ε2 were not significantly different between cases and controls. APOE ε2 carriers were observed at a frequency of 13.6% (329/2412) among cases and 15% (158/1053) among controls. Logistic regression models evaluating PD as possibly associated with ε4 or ε2 carrier status and allele dosages yielded no significant results. The mean MMSE score among all PD cases was 28.35 (SD = 2.58) and memory loss was reported in only 11.9% (105/879) of cases. Linear regression models comparing MMSE scores as predicted by ε4 or ε2 carrier status and allele dosages were not significant. CONCLUSIONS: There is no association between APOE epsilon alleles and Parkinson's disease.