Inherited Retinal Dystrophies Research Articles

Comprehensive analysis of two hotspot codons in the TUBB4B gene and associated phenotypes

Our purpose was to elucidate the genotype and ophthalmological and audiological phenotype in TUBB4B-associated inherited retinal dystrophy (IRD) and sensorineural hearing loss (SNHL), and to model the effects of all possible amino acid substitutions at the hotspot codons Arg390 and Arg391. Six patients from five families with heterozygous missense variants in TUBB4B were included in this observational study. Ophthalmological testing included best-corrected visual acuity, fundus examination, optical coherence tomography, fundus autofluorescence imaging, and full-field electroretinography (ERG). Audiological examination included pure-tone and speech audiometry in adult patients and auditory brainstem response testing in a child. Genetic testing was performed by disease gene panel analysis based on genome sequencing. The molecular consequences of the substitutions of residues 390 and 391 on TUBB4B and its interaction with α-tubulin were predicted in silico on its three-dimensional structure obtained by homology modelling. Two independent patients had amino acid exchanges at position 391 (p.(Arg391His) or p.(Arg391Cys)) of the TUBB4B protein. Both had a distinct IRD phenotype with peripheral round yellowish lesions with pigmented spots and mild or moderate SNHL, respectively. Yet the phenotype was milder with a sectorial pattern of bone spicules in one patient, likely due to a genetically confirmed mosaicism for p.(Arg391His). Three patients were heterozygous for an amino acid exchange at position 390 (p.(Arg390Gln) or p.(Arg390Trp)) and presented with another distinct retinal phenotype with well demarcated pericentral retinitis pigmentosa. All showed SNHL ranging from mild to severe. One additional patient showed a variant distinct from codon 390 or 391 (p.(Tyr310His)), and presented with congenital profound hearing loss and reduced responses in ERG. Variants at codon positions 390 and 391 were predicted to decrease the structural stability of TUBB4B and its complex with α-tubulin, as well as the complex affinity. In conclusion, the twofold larger reduction in heterodimer affinity exhibited by Arg391 substitutions suggested an association with the more severe retinal phenotype, compared to the substitution at Arg390.

Phototoxicity avoidance is a potential therapeutic approach for retinal dystrophy caused by EYS dysfunction.

Inherited retinal dystrophies (IRDs) are progressive diseases leading to vision loss. Mutation in the eyes shut homolog (EYS) gene is one of the most frequent causes of IRD. However, the mechanism of photoreceptor cell degeneration by mutant EYS has not been fully elucidated. Here, we generated retinal organoids from induced pluripotent stem cells (iPSCs) derived from patients with EYS-associated retinal dystrophy (EYS-RD). In photoreceptor cells of RD organoids, both EYS and G protein-coupled receptor kinase 7 (GRK7), one of the proteins handling phototoxicity, were not in the outer segment, where they are physiologically present. Furthermore, photoreceptor cells in RD organoids were vulnerable to light stimuli, and especially to blue light. Mislocalization of GRK7, which was also observed in eys-knockout zebrafish, was reversed by delivering control EYS into photoreceptor cells of RD organoids. These findings suggest that avoiding phototoxicity would be a potential therapeutic approach for EYS-RD.

Inherited retinal dystrophies and orphan designations in the European Union.

Inherited Retinal Dystrophies (IRD) are diverse rare diseases that affect the retina and lead to visual impairment or blindness. Research in this field is ongoing, with over 60 EU orphan medicinal products designated in this therapeutic area by the Committee for Orphan Medicinal Products (COMP) at the European Medicines Agency (EMA). Up to now, COMP has used traditional disease terms, like retinitis pigmentosa, for orphan designation regardless of the product's mechanism of action. The COMP reviewed the designation approach for IRDs taking into account all previous Orphan Designations (OD) experience in IRDs, the most relevant up to date scientific literature and input from patients and clinical experts. Following the review, the COMP decided that there should be three options available for orphan designation concerning the condition: i) an amended set of OD groups for therapies that might be used in a broad spectrum of conditions, ii) a gene-specific designation for targeted therapies, and iii) an occasional term for products that do not fit in the above two categories. The change in the approach to orphan designation in IRDs caters for different scenarios to allow an optimum approach for future OD applications including the option of a gene-specific designation. By applying this new approach, the COMP increases the regulatory clarity, efficiency, and predictability for sponsors, aligns EU regulatory tools with the latest scientific and medical developments in the field of IRDs, and ensures that all potentially treatable patients will be included in the scope of an OD.

Disease-specific variant interpretation highlighted the genetic findings in 2325 Japanese patients with retinitis pigmentosa and allied diseases

BackgroundAs gene-specific therapy for inherited retinal dystrophy (IRD) advances, unified variant interpretation across institutes is becoming increasingly important. This study aims to update the genetic findings of 86 retinitis pigmentosa...

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.

Screening copy number variations in 35 unsolved inherited retinal disease families

The purpose of this study was to screen Copy Number Variations (CNVs) in 35 unsolved Inherited Retinal Dystrophy (IRD) families. Initially, next generation sequencing, including a specific Hereditary Eye Disease Enrichment Panel or Whole exome sequencing, was employed to screen (likely) pathogenic Single-nucleotide Variants (SNVs) and small Insertions and Deletions (indels) for these cases. All available SNVs and indels were further validated and co-segregation analyses were performed in available family members by Sanger sequencing. If not, after excluding deep intronic variants, Multiplex ligation-dependent probe amplification (MLPA), quantitative fluorescence PCR (QF-PCR) and Sanger sequencing were employed to screen CNVs. We determined that 18 probands who had heterozygous SNVs/indels or whose parents were not consanguineous but had homozygous SNVs/indels in autosomal recessive IRDs genes had CNVs in another allele of these genes, 11 families had disease-causing hemizygous CNVs in X-linked IRD genes, 6 families had (likely) pathogenic heterozygous CNVs in PRPF31 gene. Of 35 families, 33 different CNVs in 16 IRD-associated genes were detected, with PRPF31, EYS and USH2A the most common disease-causing gene in CNVs. Twenty-six and 7 of them were deletion and duplication CNVs, respectively. Among them, 14 CNVs were first reported in this study. Our research indicates that CNVs contribute a lot to IRDs, and screening of CNVs substantially increases the diagnostic rate of IRD. Our results emphasize that MLPA and QF-PCR are ideal methods to validate CNVs, and the novel CNVs reported herein expand the mutational spectrums of IRDs.

Highly efficient capture approach for the identification of diverse inherited retinal disorders

Our study presents a 319-gene panel targeting inherited retinal dystrophy (IRD) genes. Through a multi-center retrospective cohort study, we validated the assay’s effectiveness and clinical utility and characterized the mutation spectrum of Taiwanese IRD patients. Between January 2018 and May 2022, 493 patients in 425 unrelated families, all initially suspected of having IRD without prior genetic diagnoses, underwent detailed ophthalmic and physical examinations (with extra-ocular features recorded) and genetic testing with our customized panel. Disease-causing variants were identified by segregation analysis and clinical interpretation, with validation via Sanger sequencing. We achieved a read depth of >200× for 94.2% of the targeted 1.2 Mb region. 68.5% (291/425) of the probands received molecular diagnoses, with 53.9% (229/425) resolved cases. Retinitis pigmentosa (RP) is the most prevalent initial clinical impression (64.2%), and 90.8% of the cohort have the five most prevalent phenotypes (RP, cone-rod syndrome, Usher’s syndrome, Leber’s congenital amaurosis, Bietti crystalline dystrophy). The most commonly mutated genes of probands that received molecular diagnosis are USH2A (13.7% of the cohort), EYS (11.3%), CYP4V2 (4.8%), ABCA4 (4.5%), RPGR (3.4%), and RP1 (3.1%), collectively accounted for 40.8% of diagnoses. We identify 87 unique unreported variants previously not associated with IRD and refine clinical diagnoses for 21 patients (7.22% of positive cases). We developed a customized gene panel and tested it on the largest Taiwanese cohort, showing that it provides excellent coverage for diverse IRD phenotypes.

Developing gene‐independent approaches for retinal dystrophies

Inherited retinal dystrophies (IRDs) display remarkable genetic heterogeneity, with over 300 retinal disease genes mapped and/or identified. Profound vision loss and blindness, often from birth, is a common occurrence in these clinically heterogeneous disease conditions. The therapeutic options for IRDs are currently very limited but recent advancements in understanding mechanisms of retinal degeneration and technological progress paved the way towards development of novel therapies. Much progress has been made in moving from gene discovery to gene therapy. However, the genetic heterogeneity of IRDs represents a significant challenge for development of gene therapy.Many studies from our group have demonstrated that the survival of cones (responsible for diurnal and high‐resolution vision) depends on the presence of rods (responsible for vision at low light levels) that secrete a diffusible trophic factor identified as rod‐derived cone viability factor (RdCVF). The discovery of RdCVF, of its receptor and mechanism of action, and the demonstration of its mutation‐independent therapeutic potential in several animal models of IRDs paved the way to the development (with the start‐up company Sparing Vision) of RdCVF neuroprotective gene therapy that entered in clinical trial in 2023.IRDs destroy photoreceptors but leave intact and functional a significant number of inner retinal cells. Using viral vectors, light‐sensitive microbial opsins can be expressed in these remaining cells, making possible their conversion into ‘artificial photoreceptors’. Our group in Paris, together with Botond Roska (IOB, Basel) and the biotech company GenSight, demonstrated that this technology, named optogenetics, can restore vision in secondary retinal neurons which can survive years after the degeneration of photoreceptors. Our groups established the foundation for the first‐in‐man clinical trial (NCT03326336) with the photoactivatable optogene ChrimsonR (developed by Ed Boyen at MIT) delivered in the eye via adeno‐associated viral vectors and electronic goggles to intensify the light and provide adaptation. We demonstrated (Nat Med 2021) that optogenetics is a remarkably effective way to restore partial vision and provide useful vision restoration in blind people.Gene‐independent approaches such as neuroprotection and optogenetics would allow for the treatment of a broad spectrum of retinal dystrophies, even at advanced stages of disease, giving hope for vision rescue and vision restoration to approximately 2 million people with IRDs worldwide.

Genetic landscape of inherited retinal dystrophies in a Welsh Tertiary Referral Centre

Aims/Purpose: Inherited retinal dystrophies (IRD) are a group of predominantly monogenic disorders which have genetically heterogeneous origins and display wide clinical phenotypic heterogeneity. The current study aims to describe the clinical diagnoses, demographics, and genetic aetiology of patients in an adult cohort of Welsh patients to provide insight into the rates of genetic diagnosis of IRD. This will help inform patient prognosis and counselling in future clinical encounters.Methods: A comprehensive database of patients attending the tertiary referral clinic from 2011 to 2022 was compiled contemporaneously. Demographic data were collated from patients' clinical records. Subjects underwent genetic screening, including targeted gene sequencing, next‐generation sequencing‐based gene panel, or whole exome sequencing, to investigate the causative mutations.Results: A total of 365 probands (191 men and 174 women). The median age at which the patients developed visual symptoms related to IRD was 42 ± 17.7 years (range, 1–86 years). Retinitis pigmentosa (176; 48.2%), Macular Dystrophy (49; 13.4%), and Cone Rod Dystrophy (41; 11.2%) were the most common clinical diagnoses in this cohort. Among all the subjects, a genetic mutation was identified in 193 (52.9%) patients and the genetic aetiology of diseases was confirmed in 147 (40.3%) patients. Pathogenic mutations were identified in 48 retinal genes with the most commonly affected genes being ABCA4, USH2A and GUCY2D with 78, 44 and 13 mutations identified overall.Conclusions: The establishment of a molecular genetic diagnosis in this group of patients serves as a basis for genetic counselling and will allow patients to enrol in current and future gene‐based clinical trials, and benefit from any future gene‐based treatments.

Multifocal electroretinogram in the diagnosis of Stargardt disease

Aims/Purpose: Electrophysiological vision tests have been a key method in the diagnosis and monitoring of inherited retinal dystrophies (IRD). With the development of mfERG, the clinical applications of these tests have expanded to cone dystrophies such as Stargardt disease, which is the most common cone‐IRD. This disease is typically associated with an autosomal recessive inheritance pattern, primarily, although not exclusively, due to mutations in the ABCA4 gene. The introduction of different diagnostic techniques such as Optical Coherence Tomography (OCT), OCT Angiography (OCTA), autofluorescence (FAF) and microperimetry has diminished the importance of fluorescein angiography (FA). These techniques have been joined by the diagnostic role of mfERG.The aim of this study is to evaluate functional changes in patients with Stargardt disease using mfERG.Methods: Eighteen eyes of patients with Stargardt disease were included. The disease was previously confirmed by genotyping with homozygous ABCA4 mutations or heterozygous PROM1 mutations. The patients were evaluated using mfERG, using the standardized international ISCEV guidelines. Electrical density and implicit time of each first‐order response (K1) in the five concentric rings centered on the fovea were measured and analysed.Results: Mean best‐corrected visual acuity (BCVA) was 0.5 (0.12–1.0). First‐order responses (K1) showed a decreased electrical density, predominantly in central rings, along with minimally prolonged latencies of the P1. FAF showed central atrophy in 6 patients, atrophy with flecks within or beyond the vascular branches in 8 and 4 patients respectively.Conclusions: Stargardt disease involves cones and is characterized by a significant decrease in foveal and central response in mfERG, even in early stages when BCVA remains relatively preserved and fundus findings are minimal. Flash ERG may show normal results or minimal abnormalities.

Whole-Exome Sequencing in Turkish Patients with Inherited Retinal Dystrophies Reveals Novel Variants in Ten Genes

Introduction: Inherited retinal dystrophies (IRDs) associated with more than 300 genes are a clinically and genetically heterogeneous group of retinal diseases. This study aimed to identify causative gene variants and molecular basis of Turkish patients with IRD. Methods: Whole-exome sequencing was performed in 28 unrelated patients. The potential pathogenicity of variants was evaluated using the American College of Medical Genetics variant interpretation guidelines, in silico prediction tools, published literature or Human Gene Mutation Database, and compatibility with inheritance patterns or known phenotypes. Results: Causative variants in 21 genes, including MERTK, SNRP200, MYO7A, AIPL1, RDH12, OTX2, ADGRV1, RPGRIP1, SPATA7, USH2A, MFSD8, CDHR1, EYS, CACNA1F, CNGA3, RDH5, TULP1, BBS2, BEST1, RS1, GUCY2D were detected in 26 (92.9%) of 28 patients. The most prevalent causative variants were observed MERTK (10.7% of cases), followed by CDHR1, AIPL1, RDH12, SPATA7, CNGA3, TULP1 (7.1% of cases, each). The most common variant type in this study was missense variants (53%), followed by frameshift (21%), nonsense (20%), and splice (6%). Twelve novel variants, 6 of frameshift and 6 of missense, were detected in ten genes. Retinitis pigmentosa was the most common phenotype followed by Leber congenital amaurosis. Conclusion: This study provides an overview of causative gene variants in Turkish patients with IRD. Variants identified in this study expand the variant spectrum of IRD genes. We believe it is essential to combine molecular and clinical data to diagnose IRD patients, especially with the emergence of therapeutic options.

The m6A reader YTHDC2 maintains visual function and retinal photoreceptor survival through modulating translation of PPEF2 and PDE6B

Inherited retinal dystrophies (IRDs) are major causes of visual impairment and irreversible blindness worldwide, while the precise molecular and genetic mechanisms are still elusive. N6-methyladenosine (m6A) modification is the most prevalent internal modification in eukaryotic mRNA. YTH domain containing 2 (YTHDC2), an m6A reader protein, has recently been identified as a key player in germline development and human cancer. However, its contribution to retinal function remains unknown. Here, we explore the role of YTHDC2 in the visual function of retinal rod photoreceptors by generating rod-specific Ythdc2 knockout mice. Results show that Ythdc2 deficiency in rods causes diminished scotopic ERG responses and progressive retinal degeneration. Multi-omics analysis further identifies Ppef2 and Pde6b as the potential targets of YTHDC2 in the retina. Specifically, via its YTH domain, YTHDC2 recognizes and binds m6A-modified Ppef2 mRNA at the coding sequence and Pde6b mRNA at the 5′-UTR, resulting in enhanced translation efficiency without affecting mRNA levels. Compromised translation efficiency of Ppef2 and Pde6b after YTHDC2 depletion ultimately leads to decreased protein levels in the retina, impaired retinal function, and progressive rod death. Collectively, our finding highlights the importance of YTHDC2 in visual function and photoreceptor survival, which provides an unreported elucidation of IRD pathogenesis via epitranscriptomics.

Retinitis Pigmentosa: Pathogenesis, Diagnostic Findings, and Treatment.

Retinitis Pigmentosa (RP) is an inherited retinal dystrophy (IRD) that causes progressive visual loss. Patients suffering from RP have a substantial influence on their everyday activities, social contacts, and jobs, lowering their quality of life. Frequent referral delays, as well as the lack of a standard therapy for the majority of patients, contribute to the significant unmet demand for RP. Any retinal injury has the potential to result in total blindness and visual impairment. Despite the fact that there is no cure for RP, people can manage it using rehabilitation programs and low-vision gadgets. The purpose of this research is to characterize the expanding treatment landscape for RP, as well as the justification for advanced therapy medicinal products (ATMPs). Vitamin A supplementscan help prevent the sluggish visual loss caused by a prevalent kind of RP. The presence of visual purple in the rods and the underlying vascular choroid causes the retina to look purplish red. The major portion of the retina damaged is the rod photoreceptor electric cell; the development of diverse diseases is progressive. Because of the retina's accessibility, immunological privilege, and compartmentalization, hereditary retinal diseases are amenable to cell and gene therapy. Therapeutic techniques that attempt to rescue photoreceptors (gene therapies) require the existence of non-functional target cells, but other therapies (cell therapies) do not require the presence of live photoreceptors. To provide successful therapy choices for RP patients at all disease phases, the development pipeline must be continually diversified and advanced, as well as ongoing efforts to encourage early patient identification and quick diagnosis. Future research will focus on avoiding vision loss in genetic eye illnesses and assisting patients in regaining their eyesight. Retinal implants, cell therapies, supplementary medications, and gene therapies may become common treatments for reducing vision loss in the future.

A Case Series of Occult Macular Dystrophy

Purpose. This case series showcases three patients with mild variation in phenotypic presentations of Occult Macular Dystrophy (OCMD), an autosomal-dominant inherited retinal dystrophy (IRD) associated with a retinitis pigmentosa-1-like-1 (RP1L1 ) gene mutation. All cases have confirmed RP1L1 gene mutations and display a range of phenotypic expressions on various imaging modalities and visual acuities. Material and Methods. Three patients of the University Eye Center (UEC) with confirmed RP1L1 gene mutations presented for follow-up or baseline imaging to rule out inherited retinal disease. All patients underwent fundus photography with and without fundus autofluorescence (FAF), optical coherence tomography (OCT), genetic testing, visual fields, and electrodiagnostic testing such as multifocal electroretinogram (mfERG) and/or full-field electroretinogram (ffERG). Results. In two of the three cases, minimal to no funduscopic changes are appreciated on ophthalmoscopy or fundus photography with and without FAF. Effects of OCMD can only be appreciated on OCT imaging, including attenuation or disruption of the photoreceptor integrity line (PIL), and cen- tral depression on visual field testing. One of the three cases displays an unusual presentation with asymmetric central outer retinal atrophy that can be appreciated on fundoscopy as well as other imaging modalities. Conclusion. Although individuals with this IRD typically funduscopically appear normal, there may be a phenotypic spectrum impacting visual potential. Diagnosis of this con- dition is heavily dependent on macular OCT, electrophysio- logical testing, and genetic testing. Keywords Occult Macular Dystrophy, RP1L1, inherited retinal disease, electroretinogram, OCT

Outcomes and Adverse Effects of Voretigene Neparvovec Treatment for Biallelic RPE65-Mediated Inherited Retinal Dystrophies in a Cohort of Patients from a Single Center.

Our study evaluated the morphological and functional outcomes, and the side effects, of voretigene neparvovec (VN) gene therapy for RPE65-mediated inherited retinal dystrophies (IRDs) in 12 eyes (six patients) at the Oxford Eye Hospital with a mean follow-up duration of 8.2 (range 1-12) months. All patients reported a subjective vision improvement 1 month after gene therapy. Best-corrected visual acuity (BCVA) remained stable (baseline: 1.28 (±0.71) vs. last follow-up: 1.46 (±0.60); p = 0.25). Average white Full-Field Stimulus Testing (FST) showed a trend towards improvement (baseline: -4.41 (±10.62) dB vs. last follow-up: -11.98 (±13.83) dB; p = 0.18). No changes in central retinal thickness or macular volume were observed. The side effects included mild intraocular inflammation (two eyes) and cataracts (four eyes). Retinal atrophy occurred in 10 eyes (eight mild, two severe) but did not impact FST measurements during the follow-up period. Increased intraocular pressure (IOP) was noted in three patients (six eyes); four eyes (two patients) required glaucoma surgery. The overall safety and effectiveness of VN treatment in our cohort align with previous VN clinical trials, except for the higher occurrence of retinal atrophy and increased IOP in our cohort. This suggests that raised IOP and retinal atrophy may be more common than previously reported.

CRB1-Associated Retinal Dystrophy Patients Have Expanded Lewis Glycoantigen-Positive T Cells.

Eye inflammation may occur in patients with inherited retinal dystrophies (IRDs) and is seen frequently in IRDs associated with mutations in the CRB1 gene. The purpose of this study was to determine the types of inflammatory cells involved in IRDs, by deep profiling the composition of peripheral blood mononuclear cells of patients with a CRB1-associated IRD. This study included 33 patients with an IRD with confirmed CRB1 mutations and 32 healthy controls. A 43-parameter flow cytometry analysis was performed on peripheral blood mononuclear cells isolated from venous blood. FlowSOM and manual Boolean combination gating were used to identify and quantify immune cell subsets. Comparing patients with controls revealed a significant increase in patients in the abundance of circulating CD4+ T cells and CD8+ T cells that express sialyl Lewis X antigen. Furthermore, we detected a decrease in plasmacytoid dendritic cells and an IgA+CD24+CD38+ transitional B-cell subset in patients with an IRD. Patients with a CRB1-associated IRD show marked changes in blood leukocyte composition, affecting lymphocyte and dendritic cell populations. These results implicate inflammatory pathways in the disease manifestations of IRDs.

The Value of Electroretinography in Identifying Candidate Genes for Inherited Retinal Dystrophies: A Diagnostic Guide.

Inherited retinal dystrophies (IRDs) are a group of heterogeneous diseases caused by genetic mutations that specifically affect the function of the rod, cone, or bipolar cells in the retina. Electroretinography (ERG) is a diagnostic tool that measures the electrical activity of the retina in response to light stimuli, and it can help to determine the function of these cells. A normal ERG response consists of two waves, the a-wave and the b-wave, which reflect the activity of the photoreceptor cells and the bipolar and Muller cells, respectively. Despite the growing availability of next-generation sequencing (NGS) technology, identifying the precise genetic mutation causing an IRD can be challenging and costly. However, certain types of IRDs present with unique ERG features that can help guide genetic testing. By combining these ERG findings with other clinical information, such as on family history and retinal imaging, physicians can effectively narrow down the list of candidate genes to be sequenced, thereby reducing the cost of genetic testing. This review article focuses on certain types of IRDs with unique ERG features. We will discuss the pathophysiology and clinical presentation of, and ERG findings on, these disorders, emphasizing the unique role ERG plays in their diagnosis and genetic testing.

ABCA4 c.6480-35A>G, a novel branchpoint variant associated with Stargardt disease.

Introduction: Inherited retinal dystrophies (IRDs) can be caused by variants in more than 280 genes. The ATP-binding cassette transporter type A4 (ABCA4) gene is one of these genes and has been linked to Stargardt disease type 1 (STGD1), fundus flavimaculatus, cone-rod dystrophy (CRD), and pan-retinal CRD. Approximately 25% of the reported ABCA4 variants affect RNA splicing. In most cases, it is necessary to perform a functional assay to determine the effect of these variants. Methods: Whole genome sequencing (WGS) was performed in one Spanish proband with Stargardt disease. The putative pathogenicity of c.6480-35A>G on splicing was investigated both in silico and in vitro. The in silico approach was based on the deep-learning tool SpliceAI. For the in vitro approach we used a midigene splice assay in HEK293T cells, based on a previously established wild-type midigene (BA29) containing ABCA4 exons 46 to 48. Results: Through the analysis of WGS data, we identified two candidate variants in ABCA4 in one proband: a previously described deletion, c.699_768+342del (p.(Gln234Phefs*5)), and a novel branchpoint variant, c.6480-35A>G. Segregation analysis confirmed that the variants were in trans. For the branchpoint variant, SpliceAI predicted an acceptor gain with a high score (0.47) at position c.6480-47. A midigene splice assay in HEK293T cells revealed the inclusion of the last 47 nucleotides of intron 47 creating a premature stop codon and allowed to categorize the variant as moderately severe. Subsequent analysis revealed the presence of this variant as a second allele besides c.1958G>A p.(Arg653His) in an additional Spanish proband in a large cohort of IRD cases. Conclusion: A splice-altering effect of the branchpoint variant, confirmed by the midigene splice assay, along with the identification of this variant in a second unrelated individual affected with STGD, provides sufficient evidence to classify the variant as likely pathogenic. In addition, this research highlights the importance of studying non-coding regions and performing functional assays to provide a conclusive molecular diagnosis.

Progression of Rare Inherited Retinal Dystrophies May Be Monitored by Adaptive Optics Imaging.

Inherited retinal dystrophies (IRDs) are bilateral genetic conditions of the retina, leading to irreversible vision loss. This study included 55 eyes afflicted with IRDs affecting the macula. The diseases examined encompassed Stargardt disease (STGD), cone dystrophy (CD), and cone-rod dystrophy (CRD) using adaptive optics (Rtx1™; Imagine Eyes, Orsay, France). Adaptive optics facilitate high-quality visualisation of retinal microstructures, including cones. Cone parameters, such as cone density (DM), cone spacing (SM), and regularity (REG), were analysed. The best corrected visual acuity (BCVA) was assessed as well. Examinations were performed twice over a 6-year observation period. A significant change was observed in DM (1282.73/mm2 vs. 10,073.42/mm2, p< 0.001) and SM (9.83 μm vs. 12.16 μm, p< 0.001) during the follow-up. BCVA deterioration was also significant (0.16 vs. 0.12, p = 0.001), albeit uncorrelated with the change in cone parameters. No significant difference in REG was detected between the initial examination and the follow-up (p = 0.089).

Molecular genetic diagnostics for inherited retinal dystrophies in the clinical setting

ObjectiveTo evaluate the success of diagnostic genetic testing in inherited retinal dystrophy (IRD) patients in the clinical setting. DesignRetrospective cohort analysis. ParticipantsA total of 446 consecutive participants from diverse ethnic backgrounds living in western Canada. MethodsClinical information was collected from participants, including family history, and they underwent a full ophthalmic examination with chart review. Those with a suspected IRD were offered panel-based genetic testing of 351 genes between March 1, 2019, and February 28, 2022. The main outcome measure was effect of the genetic testing results on clinical diagnosis. ResultsGenetic testing established a conclusive molecular diagnosis in 249 of 446 cases (55.8%), a clearly negative result in 90 of 446 cases (20.1%), and an inconclusive diagnosis in 108 of 446 cases (24.2%). Conclusive disease-causing variants were identified in 69 genes, and the most commonly affected genes were ABCA4 (31 variants), USH2A (25 variants), and RPGR (19 variants). The inconclusive group included likely novel autosomal dominant variants or a pathogenic variant with a variant of uncertain significance in the same gene for a recessive phenotype. Notably, an inconclusive molecular genetic diagnosis was seen in as many as 47.3% of East Asian participants with an outer retinal dystrophy. ConclusionsThis study represents the largest review of molecular genetic testing in IRDs in Canada. That negative or inconclusive results obtained in approximately 45% of cases demonstrates that there is an important need for new research into molecular genetic causes of IRDs. This is particularly true in addressing the problem of interpreting a variant of uncertain significance in ethnic minorities.