Analysis of PDE6G mutations in a patient with retinitis pigmentosa.

BACKGROUND: Mutations in PDE6A and PDE6B are known to cause autosomal recessive RP in humans, On the other hand, mutations in PDE6G are rare but can lead to severe early-onset RP. CASE PRESENTATION: An 8-year-old Chinese boy was referred to our hospital for poor vision issues. Refraction with cycloplegia showed high hyperopia with astigmatism both eyes. Funduscopic examination revealed typical bone spicule-type pigment deposits in the periphery and midperiphery. The patient was given glasses and a whole exome sequencing containing mitochondrial genes was performed. The results of genetic testing showed that there was a heterozygous frameshift mutation and a segment deletion in the proband's PDE6G gene. Analysis of the parental genes showed that frameshift mutation was inherited from the proband's mother and segment deletion from his father. CONCLUSIONS: In this paper, we give a firsthand report that the complex heterozygous mutations of PDE6G gene can causes autosomal recessiveRP (arRP), which expands the understanding of the pathogenic genes of RP.

Clinical characterizations and molecular genetic study of two co-segregating variants in PDZD7 and PDE6C genes leading simultaneously to non-syndromic hearing loss and achromatopsia.

BACKGROUND: Autosomal recessive non-syndromic hearing loss (NSHL) and cone dystrophies (CODs) are highly genetically and phenotypically heterogeneous disorders. In this study, we applied the whole exome sequencing (WES) to find the cause of HL and COD in an Iranian consanguineous family with three affected individuals. METHODS: Three members from an Iranian consanguineous family who were suffering from NSHL and visual impairment were ascertained in this study. Comprehensive clinical evaluations and genetic analysis followed by bioinformatic and co-segregation studies were performed to diagnose the cause of these phenotypes. Data were collected from 2020 to 2022. RESULTS: All cases showed congenital bilateral NSHL, decreased visual acuity, poor color discrimination, photophobia and macular atrophy. Moreover, cornea, iris and anterior vitreous were within normal limit in both eyes, decreased foveal sensitivity, central scotoma and generalized depression of visual field were seen in three cases. WES results showed two variants, a novel null variant (p.Trp548Ter) in the PDE6C gene causing COD type 4 (Achromatopsia) and a previously reported variant (p.Ile84Thr) in the PDZD7 gene causing NSHL. Both variants were found in the cis configuration on chromosome 10 with a genetic distance of about 8.3 cM, leading to their co-inheritance. However, two diseases could appear independently in subsequent generations due to crossover during meiosis. CONCLUSIONS: Here, we could successfully determine the etiology of a seemingly complex phenotype in two adjacent genes. We identified a novel variant in the PDE6C gene, related to achromatopsia. Interestingly, this variant could cooperatively cause visual disorders: cone dystrophy and cone-rod dystrophy.

A combination treatment based on drug repurposing demonstrates mutation-agnostic efficacy in pre-clinical retinopathy models.

Inherited retinopathies are devastating diseases that in most cases lack treatment options. Disease-modifying therapies that mitigate pathophysiology regardless of the underlying genetic lesion are desirable due to the diversity of mutations found in such diseases. We tested a systems pharmacology-based strategy that suppresses intracellular cAMP and Ca2+ activity via G protein-coupled receptor (GPCR) modulation using tamsulosin, metoprolol, and bromocriptine coadministration. The treatment improves cone photoreceptor function and slows degeneration in Pde6ßrd10 and RhoP23H/WT retinitis pigmentosa mice. Cone degeneration is modestly mitigated after a 7-month-long drug infusion in PDE6A-/- dogs. The treatment also improves rod pathway function in an Rpe65-/- mouse model of Leber congenital amaurosis but does not protect from cone degeneration. RNA-sequencing analyses indicate improved metabolic function in drug-treated Rpe65-/- and rd10 mice. Our data show that catecholaminergic GPCR drug combinations that modify second messenger levels via multiple receptor actions provide a potential disease-modifying therapy against retinal degeneration.

A New Mouse Model for Usher Syndrome Crossing Kunming Mice with CBA/J Mice.

BACKGROUND: Previously, we discovered a strain of Kunming mice, referred to as the KMush/ush strain, that exhibited notably abnormal electroretinogram (ERG) readings and elevated thresholds for auditory brainstem responses (ABRs), which resembled the characteristics of Usher Syndrome (USH). We successfully identified the pathogenic genes, Pde6b and Adgrv1, after KMush/ush crossbred with CBA/CaJ mice, referred to as CBA-1ush/ush, CBA-2ush/ush or CBA-2ush/ush. In this investigation, we crossbred KMush/ush and CBA/J mice to establish novel recombinant inbred lines and analysed their phenotypic and genotypic characteristics. METHODS: ERG readings, ABR testing, fundus morphology, histological examination of the retina and inner ear, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis, western blotting, DNA sequence analysis and behavioural experiments were performed to assess the phenotypes and genotypes of the progeny lines. RESULTS: No obvious waveforms in the ERG were detected in F1 hybrid mice while normal ABR results were recorded. The F2 hybrids, which were called J1ush/ush or J2ush/ush, exhibited segregated hearing-loss phenotypes. J1ush/ush mice had a retinitis pigmentosa (RP) phenotype with elevated ABR thresholds, whereas J2ush/ush mice exhibited only the RP phenotype. Interestingly, J1ush/ush mice showed significantly higher ABR thresholds than wild-type mice at 28 days post born (P28), and RT-qPCR and DNA-sequencing analysis showed that Adgrv1 gene expression was significantly altered in J1ush/ush mice, but histological analysis showed no significant structural changes in the organ of Corti or spiral ganglia. Further elevation of ABR-related hearing thresholds by P56 manifested only as a reduced density of spiral ganglion cells, which differed significantly from the previous pattern of cochlear alterations in CBA-2ush/ush mice. CONCLUSIONS: We successfully introduced the hearing-loss phenotype of inbred mice with USH into CBA/J mice, which provides a good animal model for future studies on the important physiological roles of the Adgrv1 gene in inner-ear structure and for therapeutic studies targeting Adgrv1-mutated USH.

Genetics, Clinical Characteristics, and Natural History of PDE6B-Associated Retinal Dystrophy.

PURPOSE: To analyze the clinical characteristics, natural history, and genetics of PDE6B-associated retinal dystrophy. DESIGN: Retrospective, observational cohort study. METHODS: Review of medical records and retinal imaging, including fundus autofluorescence (FAF) imaging and spectral-domain optical coherence tomography (SD-OCT) of patients with molecularly confirmed PDE6B-associated retinal dystrophy in a single tertiary referral center. Genetic results were reviewed, and the detected variants were assessed. RESULTS: Forty patients (80 eyes) were identified and evaluated longitudinally. The mean age (±SD, range) was 42.1 years (± 19.0, 10-86) at baseline, with a mean follow-up time of 5.2 years. Twenty-nine (72.5%) and 27 (67.5%) patients had no or mild visual acuity impairment at baseline and last visit, respectively. Best-corrected visual acuity (BCVA) was 0.56 ± 0.72 LogMAR (range -0.12 to 2.80) at baseline and 0.63 ± 0.73 LogMAR (range 0.0-2.80) at the last visit. BCVA was symmetrical in 87.5% of patients. A hyperautofluorescent ring was observed on FAF in 48 and 46 eyes at baseline and follow-up visit, respectively, with a mean area of 7.11 ± 4.13 mm2 at baseline and mean of 6.13 ± 3.62 mm2 at the follow-up visit. Mean horizontal ellipsoid zone width at baseline was 1946.1 ± 917.2 µm, which decreased to 1763.9 ± 827.9 µm at follow-up. Forty-four eyes had cystoid macular edema at baseline (55%), and 41 eyes (51.3%) at follow-up. There were statistically significant changes during the follow-up period in terms of BCVA and the ellipsoid zone width. Genetic analysis identified 43 variants in the PDE6B gene, including 16 novel variants. CONCLUSIONS: This study details the natural history of PDE6B-retinopathy in the largest cohort to date. Most patients had mild to no BCVA loss, with slowly progressive disease, based on FAF and OCT metrics. There is a high degree of disease symmetry and a wide window for intervention.

Phenotypic characteristics of Danish patients with achromatopsia.

PURPOSE: To describe the phenotype of Danish patients with genetically verified achromatopsia (ACHM) with special focus on signs of progression on structural or functional parameters, and possible genotype-phenotype correlations. METHODS: Forty-eight patients were identified, with disease-causing variants in five different genes: CNGA3, CNGB3, GNAT2, PDE6C and PDE6H. Longitudinal evaluation was possible for 11 patients and 27 patients participated in a renewed in-depth phenotyping consisting of visual acuity assessment, optical coherence tomography (OCT), fundus autofluorescence, colour vision evaluation, contrast sensitivity, mesopic microperimetry and full-field electroretinography. Foveal morphology was evaluated based on OCT images for all 48 patients using a grading system based on the integrity of the hyperreflective photoreceptor band, the inner segment ellipsoid zone (ISe). Signs of progression were evaluated based on longitudinal data and correlation with age. RESULTS: We found a statistically significant positive correlation between OCT grade and age (Spearman ρ = 0.62, p < 0.0001) and we observed changes in the foveal morphology in 2 of 11 patients with ≥5 years of follow-up. We did not find any convincing correlation between age and functional parameters (visual acuity, retinal sensitivity and contrast sensitivity) nor did we find correlation between structural and functional parameters, or any clear genotype-phenotype correlation. CONCLUSIONS: Some patients with ACHM demonstrate signs of progressive foveal changes in OCT characteristics with increasing age. This is relevant in terms of possible new treatments. However, functional characteristics, such as visual acuity, remained stable despite changing foveal structure. Thus, seen from a patient perspective, ACHM can still be considered a non-progressive condition.

A systematic review of inherited retinal dystrophies in Pakistan: updates from 1999 to April 2023.

BACKGROUND: Inherited retinal degenerations (IRDs) are a group of rare genetic conditions affecting retina of the eye that range in prevalence from 1 in 2000 to 1 in 4000 people globally. This review is based on a retrospective analysis of research articles reporting IRDs associated genetic findings in Pakistani families between 1999 and April 2023. METHODS: Articles were retrieved through survey of online sources, notably, PubMed, Google Scholar, and Web of Science. Following a stringent selection criterion, a total of 126 research articles and conference abstracts were considered. All reported variants were cross-checked and validated for their correct genomic nomenclature using different online resources/databases, and their pathogenicity scores were explained as per ACMG guidelines. RESULTS: A total of 277 unique sequence variants in 87 distinct genes, previously known to cause IRDs, were uncovered. In around 70% cases, parents of the index patient were consanguineously married, and approximately 88.81% of the detected variants were found in a homozygous state. Overall, more than 95% of the IRDs cases were recessively inherited. Missense variants were predominant (41.88%), followed by Indels/frameshift (26.35%), nonsense (19.13%), splice site (12.27%) and synonymous change (0.36%). Non-syndromic IRDs were significantly higher than syndromic IRDs (77.32% vs. 22.68%). Retinitis pigmentosa (RP) was the most frequently observed IRD followed by Leber's congenital amaurosis (LCA). Altogether, mutations in PDE6A gene was the leading cause of IRDs in Pakistani families followed by mutations in TULP1 gene. CONCLUSION: In summary, Pakistani families are notable in expressing recessively inherited monogenic disorders including IRDs likely due to the highest prevalence of consanguinity in the country that leads to expression of rare pathogenic variants in homozygous state.

Establishment and visual analysis of CBA/J-Pde6bY347Y/Y347X and C3H/HeJ-Pde6bY347Y/Y347X mice.

In CBA/J and C3H/HeJ mice, retinitis pigmentosa is inherited as an autosomal-recessive trait due to a mutation in Pde6b, which encodes cGMP phosphodiesterase subunit b. In these strains, the Y347X mutation in Pde6b leads to the upregulation of cGMP levels, increased Ca2+ influx induces rod death, and the outer segment and rod cells entirely disappeared by 35 days after birth. In the present study, we utilized the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) 9-mediated gene editing to repair the Y347X mutation in CBA/J and C3H/HeJ mice. Evaluation of the established CBA/J-Pde6bY347Y/Y347X and C3H/HeJ-Pde6bY347Y/Y347X mice, which were confirmed to have normal retinal layers by live fundoscopic imaging and histopathological analysis, revealed improved visual acuity based on the visual cliff and light/dark latency tests. Furthermore, our analyses revealed that the visible platform test was a more effective tool for testing visual behavior in these mice. The results suggest that the established strains can serve as control groups for CBA/J and C3H/HeJ in ophthalmology studies involving retinitis pigmentosa.

Reconstitution of the phosphodiesterase 6 maturation process important for photoreceptor cell function.

The sixth family phosphodiesterases (PDE6) are principal effector enzymes of the phototransduction cascade in rods and cones. Maturation of nascent PDE6 protein into a functional enzyme relies on a coordinated action of ubiquitous chaperone HSP90, its specialized cochaperone aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1), and the regulatory Pγ-subunit of PDE6. Deficits in PDE6 maturation and function underlie severe visual disorders and blindness. Here, to elucidate the roles of HSP90, AIPL1, and Pγ in the maturation process, we developed the heterologous expression system of human cone PDE6C in insect cells allowing characterization of the purified enzyme. We demonstrate that in the absence of Pγ, HSP90, and AIPL1 convert the inactive and aggregating PDE6C species into dimeric PDE6C that is predominantly misassembled. Nonetheless, a small fraction of PDE6C is properly assembled and fully functional. From the analysis of mutant mice that lack both rod Pγ and PDE6C, we conclude that, in contrast to the cone enzyme, no maturation of rod PDE6AB occurs in the absence of Pγ. Co-expression of PDE6C with AIPL1 and Pγ in insect cells leads to a fully mature enzyme that is equivalent to retinal PDE6. Lastly, using immature PDE6C and purified chaperone components, we reconstituted the process of the client maturation in vitro. Based on this analysis we propose a scheme for the PDE6 maturation process.

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.

Rod and Cone Function Measured Objectively by Chromatic Pupil Campimetry Show a Different Preservation Between Distinct Genotypes in Retinitis Pigmentosa.

Purpose: Verifying whether specific genotypes causing retinitis pigmentosa (RP) show differences in the preservation of rod and cone function measured by chromatic pupil campimetry (CPC). Methods: Sixty-three RP eyes (37 male, 14-58 years) were measured using CPC with specific photopic and scotopic protocols, and the relative maximal constriction amplitudes and latencies to constriction onset were analyzed per genotype (RP due to variants in EYS, n = 14; PDE6A, n = 10; RPE65, n = 15; USH2A, n = 10; and RPGR, n = 14). Correlation analyses between the pupillary responses were performed with age, full-field stimulus threshold (FST), and optical coherence tomography (OCT) for cones and rods, respectively, to the genotype. Results: Pupillary responses were most severely reduced in RPE65-RP. Patients with disease-associated variants in EYS and USH2A were accompanied with better-preserved rod function compared with the other subgroups, reaching statistical significance between EYS and RPE65. Cone function was statistically significantly correlated with age in USH2A-RP with an annual decline of 2.4%. Correlations of pupillary responses were found with FST but barely with the ellipsoid zone area in OCT. Latency was significantly more prolonged in RPE65-RP compared with the other genotypes for cones. Conclusions: Rod and cone function measured objectively by CPC showed a different preservation between genotypes in RP. However, heterogeneity inside the same genotype was present. CPC data correlated with FST, but structural OCT parameters seem to be limited indicators for photoreceptor function in RP. Prolonged time dynamics for cones in RPE65 mutations suggest an impact on cone processing and might provide additional information in the evaluation of therapy effects.

Prenylation is essential for the enrichment of cone phosphodiesterase-6 (PDE6) in outer segments and efficient cone phototransduction.

Phosphodiesterase-6 (PDE6) is the key phototransduction effector enzyme residing in the outer segment (OS) of photoreceptors. Cone PDE6 is a tetrameric protein consisting of two inhibitory subunits (γ') and two catalytic subunits (α'). The catalytic subunit of cone PDE6 contains a C-terminus prenylation motif. Deletion of PDE6α' C-terminal prenylation motif is linked to achromatopsia (ACHM), a type of color blindness in humans. However, mechanisms behind the disease and roles for lipidation of cone PDE6 in vision are unknown. In this study, we generated two knock-in mouse models expressing mutant variants of cone PDE6α' lacking the prenylation motif (PDE6α'∆C). We find that the C-terminal prenylation motif is the primary determinant for the association of cone PDE6 protein with membranes. Cones from PDE6α'∆C homozygous mice are less sensitive to light, and their response to light is delayed, whereas cone function in heterozygous PDE6α'∆C/+ mice is unaffected. Surprisingly, the expression level and assembly of cone PDE6 protein were unaltered in the absence of prenylation. Unprenylated assembled cone PDE6 in PDE6α'∆C homozygous animals is mislocalized and enriched in the cone inner segment and synaptic terminal. Interestingly, the disk density and the overall length of cone OS in PDE6α'∆C homozygous mutants are altered, highlighting a novel structural role for PDE6 in maintaining cone OS length and morphology. The survival of cones in the ACHM model generated in this study bodes well for gene therapy as a treatment option for restoring vision in patients with similar mutations in the PDE6C gene.

Next-generation sequencing--based genetic testing and phenotype correlation in retinitis pigmentosa patients from India.

Purpose: Inherited retinal dystrophies (IRD) are a heterogeneous group of retinal diseases leading to progressive loss of photoreceptors through apoptosis. Retinitis pigmentosa (RP) is considered the most common form of IRD. Panel-based testing in RP has proven effective in identifying the causative genetic mutations in 70% and 80% of the patients. This is a retrospective, observational, single-center study of 107 RP patients who had undergone next-generation sequencing-based targeted gene panel testing for IRD genes. These patients were inspected for common phenotypic features to arrive at meaningful genotype-phenotype correlation. Methods: Patients underwent complete ophthalmic examination, and blood was collected from the proband for DNA extraction after documenting the pedigree. Targeted Next Generation Sequencing (NGS) was done by panel-based testing for IRD genes followed by co-segregation analysis wherever applicable. Results: Of the 107 patients, 72 patients had pathogenic mutations. The mean age of onset of symptoms was 14 ± 12 years (range: 5-55). Mean (Best Corrected Visual Acuity) BCVA was 6/48 (0.9 logMAR) (range 0.0-3.0). At presentation, over one-third of eyes had BCVA worse than 6/60 (<1 logMAR). Phenotype analysis with the gene defects showed overlapping features, such as peripheral well-defined chorioretinal atrophic patches in patients with CERKL, PROM1, and RPE65 gene mutations and large macular lesions in patients with RDH12 and CRX gene mutations, respectively. Nummular or clump-like pigmentation was noted in CRB1, TTC8, PDE6A, and PDE6B. Conclusion: NGS-based genetic testing can help clinicians to diagnose RP more accurately, and phenotypic correlations can also help in better patient counselling with respect to prognosis and guidance regarding ongoing newer gene-based therapies.

HSP90α is needed for the survival of rod photoreceptors and regulates the expression of rod PDE6 subunits.

Heat shock protein 90 (HSP90) is an abundant molecular chaperone that regulates the stability of a small set of proteins essential in various cellular pathways. Cytosolic HSP90 has two closely related paralogs: HSP90α and HSP90ß. Due to the structural and sequence similarities of cytosolic HSP90 paralogs, identifying the unique functions and substrates in the cell remains challenging. In this article, we assessed the role of HSP90α in the retina using a novel HSP90α murine knockout model. Our findings show that HSP90α is essential for rod photoreceptor function but was dispensable in cone photoreceptors. In the absence of HSP90α, photoreceptors developed normally. We observed rod dysfunction in HSP90α knockout at 2 months with the accumulation of vacuolar structures, apoptotic nuclei, and abnormalities in the outer segments. The decline in rod function was accompanied by progressive degeneration of rod photoreceptors that was complete at 6 months. The deterioration in cone function and health was a "bystander effect" that followed the degeneration of rods. Tandem mass tag proteomics showed that HSP90α regulates the expression levels of <1% of the retinal proteome. More importantly, HSP90α was vital in maintaining rod PDE6 and AIPL1 cochaperone levels in rod photoreceptor cells. Interestingly, cone PDE6 levels were unaffected. The robust expression of HSP90ß paralog in cones likely compensates for the loss of HSP90α. Overall, our study demonstrated the critical need for HSP90α chaperone in the maintenance of rod photoreceptors and showed potential substrates regulated by HSP90α in the retina.

A Novel Intronic Deletion in PDE6B Causes Autosomal Recessive Retinitis Pigmentosa by Interfering with RNA Splicing.

INTRODUCTION: Retinitis pigmentosa (RP) is a rare degenerative retinal disease caused by mutations in approximately seventy genes. Currently, despite the availability of large-scale DNA sequencing technologies, ∼30-40% of patients still cannot be diagnosed at the molecular level. In this study, we investigated a novel intronic deletion of PDE6B, encoding the beta subunit of phosphodiesterase 6 in association with recessive RP. METHODS: Three unrelated consanguineous families were recruited from the northwestern part of Pakistan. Whole exome sequencing was performed for the proband of each family, and the data were analyzed according to an in-house computer pipeline. Relevant DNA variants in all available members of these families were assessed through Sanger sequencing. A minigene-based splicing assay was also performed. RESULTS: The clinical phenotype for all patients was compatible with rod cone degeneration, with the onset during childhood. Whole exome sequencing revealed a homozygous 18 bp intronic deletion (NM_000283.3:c.1921-20_1921-3del) in PDE6B, which co-segregated with disease in 10 affected individuals. In vitro splicing tests showed that this deletion causes aberrant RNA splicing of the gene, leading to the in-frame deletion of 6 codons and, likely, to disease. CONCLUSION: Our findings further expand the mutational spectrum of the PDE6B gene.

A novel homozygous missense substitution p.Thr313Ile in the PDE6B gene underlies autosomal recessive retinitis pigmentosa in a consanguineous Pakistani family.

BACKGROUND: Retinitis pigmentosa (RP) is one of the most frequent hereditary retinal diseases that often starts with night blindness and eventually leads to legal blindness. Our study aimed to identify the underlying genetic cause of autosomal recessive retinitis pigmentosa (arRP) in a consanguineous Pakistani family. METHODS: Following a detailed ophthalmological examination of the patients by an ophthalmologist, whole-exome sequencing was performed on the proband's DNA to delineate the genetic cause of RP in the family. In-depth computational methods, in-silico analysis, and familial co-segregation study were performed for variant detection and validation. RESULTS: We studied an inbred Pakistani family with two siblings affected by retinitis pigmentosa. The proband, a 32 years old female, was clinically diagnosed with RP at the age of 6 years. A classical night blindness symptom was reported in the proband since her early childhood. OCT report showed a major reduction in the outer nuclear layer and the ellipsoid zone width, leading to the progression of the disease. Exome sequencing revealed a novel homozygous missense mutation (c.938C > T;p.Thr313Ile) in exon 12 of the PDE6B gene. The mutation p.Thr313Ile co-segregated with RP phenotype in the family. The altered residue (p.Thr313) was super conserved evolutionarily across different vertebrate species, and all available in silico tools classified the mutation as highly pathogenic. CONCLUSION: We present a novel homozygous pathogenic mutation in the PDE6B gene as the underlying cause of arRP in a consanguineous Pakistani family. Our findings highlight the importance of missense mutations in the PDE6B gene and expand the known mutational repertoire of PDE6B-related RP.

A new mutation in the PDE6C gene in achromatopsia.

PURPOSE: To report a case of achromatopsia with a new mutation in the PDE6C gene which has not been previously described. METHODS: Case report. PATIENTS: A single patient. RESULTS: A 35-year-old woman with poor vision and impaired color vision. Fundus examination of both eyes (OU) revealed small optic discs. Optical coherence tomography (OCT) showed photoreceptor segment defects and a disruption of the ellipsoid layer in the foveal region, with intact overlying outer limiting membrane and underlying RPE bands. The electroretinogram (ERG) showed scotopic responses: DA 0.01: normal amplitude, b-wave latency at upper limit of normal / slightly increased. DA 3 and DA 10: slightly increased b-wave latency, asymmetry in b amplitude, being lower in the left eye. Oscillatory potentials: no responses are obtained. Photopic responses: LA-3: greatly increased latencies, decreased amplitude. Subsequently, a case of incomplete achromatopsia was suspected. Therefore, a genetic study was carried out showing the homozygous presence of the undescribed pathogenic variant c.660_661del (p.Ser221Tyrfs * 15) in exon 3 of the PDE6C gene. CONCLUSION: Achromatopsia is an autosomal-recessive genetic disease characterized by decreased visual acuity, color blindness, photophobia, and nystagmus. Due to the variability of genetic mutations in achromatopsia, genetic characterization is mandatory in order to improve the efficiency in molecular diagnosis. This data may be useful in future therapeutic strategies. We present a previously undescribed mutation in the PDE6C gene in a patient with incomplete achromatopsia.

Characterization of a novel Pde6b-deficient rat model of retinal degeneration and treatment with adeno-associated virus (AAV) gene therapy.

In humans, mutations in the beta subunit of cGMP-phosphodiesterase type 6 (PDE6B) cause autosomal recessive retinitis pigmentosa (RP), which typically has an aggressive clinical course of early-onset severe vision loss due to rapid photoreceptor degeneration. In this study, we describe the generation of a novel Pde6b-deficient rat model using CRISPR-Cas9 genome editing. We characterize the model at multiple time points using clinical imaging modalities as well as histology with immunohistochemistry to show rapid photoreceptor degeneration compared to wild-type and heterozygous animals. We describe the manufacture of two different adeno-associated viral (AAV) vectors (AAV2/1, AAV2/5) under current Good Manufacturing Practices (cGMP) and demonstrate their ability to drive human PDE6B expression in vivo. We further demonstrate the ability of AAV-mediated subretinal gene therapy to delay photoreceptor loss in Pde6b-deficient rats compared to untreated controls. However, severe progressive photoreceptor loss was noted even in treated eyes, likely due to the aggressive nature of the disease. These data provide useful preclinical data to guide the development of potential human gene therapy for PDE6B-associated RP. In addition, the rapid photoreceptor degeneration of the Pde6b-deficient rat with intact inner retina may provide a useful model for the study of cell replacement strategies.

Chromatic Pupillometry as a Putative Screening Tool for Heritable Retinal Disease in Rhesus Macaques.

Purpose: Non-human primates (NHPs) are useful models for human retinal disease. Chromatic pupillometry has been proposed as a noninvasive method of identifying inherited retinal diseases (IRDs) in humans; however, standard protocols employ time-consuming dark adaptation. We utilized shortened and standard dark-adaptation protocols to compare pupillary light reflex characteristics following chromatic stimulation in rhesus macaques with achromatopsia to wild-type (WT) controls with normal retinal function. Methods: Nine rhesus macaques homozygous for the p.R656Q mutation (PDE6C HOMs) and nine WT controls were evaluated using chromatic pupillometry following 1-minute versus standard 20-minute dark adaptations. The following outcomes were measured and compared between groups: pupil constriction latency, peak constriction, pupil constriction time, and constriction velocity. Results: Pupil constriction latency was significantly longer in PDE6C HOMs with red-light (P = 0.0002) and blue-light (P = 0.04) stimulation versus WT controls. Peak constriction was significantly less in PDE6C HOMs with all light stimulation compared to WT controls (P < 0.0001). Pupil constriction time was significantly shorter in PDE6C HOMs versus WT controls with red-light (P = 0.04) and white-light (P = 0.003) stimulation. Pupil constriction velocity was significantly slower in PDE6C HOMs versus WT controls with red-light (P < 0.0001), blue-light (P < 0.0001), and white-light (P = 0.0002) stimulation. Dark adaptation time only significantly affected peak (P = 0.008) and time of pupil constriction (P = 0.02) following blue-light stimulation. Conclusions: Chromatic pupillometry following 1- and 20-minute dark adaptation is an effective tool for screening NHPs for achromatopsia. Translational Relevance: Rapid identification of NHPs with IRDs will provide animal research models to advance research and treatment of achromatopia in humans.

Elevated retinal cGMP is not associated with elevated circulating cGMP levels in a canine model of retinitis pigmentosa.

PURPOSE: To investigate whether raised levels of retinal cyclic guanosine monophosphate (cGMP) was reflected in plasma levels in PDE6A-/- dogs. MATERIALS AND METHODS: Retina was collected from 2-month-old wildtype dogs (PDE6A+/+, N = 6), heterozygous dogs (PDE6A+/-, N = 4) and affected dogs (PDE6A-/-, N = 3) and plasma was collected from 2-month-old wildtype dogs (PDE6A+/+, N = 5), heterozygous dogs (PDE6A+/-, N = 5) and affected dogs (PDE6A-/-, N = 5). Retina and plasma samples were measured by ELISA. RESULTS: cGMP levels in retinal samples of PDE6A-/- dogs at 2 months of age were significantly elevated. There was no significant difference in plasma cGMP levels between wildtype and PDE6A-/- or PDE6A+/- puppies. However, the plasma cGMP levels of the PDE6A-/- puppies were significantly lower than that of PDE6A+/- puppies. CONCLUSION: cGMP levels in the plasma from PDE6A-/- was not elevated when compared to control dogs. At the 2-month timepoint, cGMP plasma levels would not be a useful biomarker for disease.