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 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.

Does Background Matter? A Comparative Characterization of Mouse Models of Autosomal Retinitis Pigmentosa rd1 and Pde6b-KO.

Many retinal degenerative diseases result in vision impairment or permanent blindness due to photoreceptor loss or dysfunction. It has been observed that Pde6brd1 mice (rd1), which carry a spontaneous nonsense mutation in the pde6b gene, have a strong phenotypic similarity to patients suffering from autosomal recessive retinitis pigmentosa. In this study, we present a novel mouse model of retinitis pigmentosa generated through pde6b gene knockout using CRISPR/Cas9 technology. We compare this Pde6b-KO mouse model to the rd1 mouse model to gain insights into the progression of retinal degeneration. The functional assessment of the mouse retina and the tracking of degeneration dynamics were performed using electrophysiological methods, while retinal morphology was analyzed through histology techniques. Interestingly, the Pde6b-KO mouse model demonstrated a higher amplitude of photoresponse than the rd1 model of the same age. At postnatal day 12, the thickness of the photoreceptor layer in both mouse models did not significantly differ from that of control animals; however, by day 15, a substantial reduction was observed. Notably, the decline in the number of photoreceptors in the rd1 model occurred at a significantly faster rate. These findings suggest that the C3H background may play a significant role in the early stages of retinal degeneration.

Quantitative and qualitative characterization of retinal dystrophies in canine models of inherited retinal diseases using spectral domain optical coherence tomography (SD-OCT).

The purpose of this study was to establish spectral domain optical coherence tomography (SD-OCT) assessment data in well-established canine models of inherited retinal dystrophies: PDE6B-rod-cone dysplasia 1 (RCD1: early onset retinitis pigmentosa), PRCD-progressive rod-cone degeneration (PRCD: late onset retinitis pigmentosa), CNGB3-achromatopsia, and RPE65-Leber congenital amaurosis (LCA). High resolution SD-OCT images of the retina were acquired from both eyes in 5 planes: temporal; superotemporal; superior; nasal; and inferior in adult dogs with: RCD1 (n = 4 dogs, median age: 1.5 yrs); PRCD (n = 2, 4.3 yrs); LCA (n = 3, 5.2 yrs); achromatopsia (n = 3, 4.2 yrs); and wild types (wt, n = 6, 5.5 yrs). Total, inner and outer retinal thicknesses and ellipsoid zone were analyzed. In selected animals, histomorphometric evaluations were performed. In dogs with RCD1, PRCD, and LCA, the thickness of the outer retina was, compared to wt, significantly decreased (p ≤ 0.02) in all OCT imaging planes, and in superotemporal and inferior imaging planes in dogs with achromatopsia. No significant thinning was observed in inner retina thickness in any disease model except in the inferior imaging plane in dogs with RCD1. Dogs with RCD1, PRCD, and LCA had significantly more areas with disrupted ellipsoid zone in the presumed area centralis than wt (p ≤ 0.001). OCT findings provide baseline information for research of retinal dystrophies using these canine models.

Novel variants in PDE6A and PDE6B genes and its phenotypes in patients with retinitis pigmentosa in Chinese families.

BACKGROUND: Retinitis pigmentosa (RP) is a genetically heterogeneous disease with 89 causative genes identified to date. However, only approximately 60% of RP cases genetically solved to date, predicating that many novel disease-causing variants are yet to be identified. The purpose of this study is to identify novel variants in PDE6A and PDE6B genes and present its phenotypes in patients with retinitis pigmentosa in Chinese families. METHODS: Five retinitis pigmentosa patients with PDE6A variants and three with PDE6B variants were identified through a hereditary eye disease enrichment panel (HEDEP), all patients' medical and ophthalmic histories were collected, and ophthalmological examinations were performed, followed by an analysis of the possible causative variants. Sanger sequencing was used to verify the variants. RESULTS: We identified 20 variants in eight patients: 16 of them were identified in either PDE6A or PDE6B in a compound heterozygous state. Additional four heterozygous variants were identified in the genes ADGRA3, CA4, OPTN, RHO. Two novel genetic changes in PDE6A were identified (c.1246G > A and c.1747 T > A), three novel genetic changes in PDE6B were identified (c.401 T > C, c.2293G > C and c.1610-1612del), out of the novel identified variants one was most probably non-pathogenic (c.2293G > C), all other novel variants are pathogenic. Additional variant was identified in CA4 and RHO, which can cause ADRP (c.243G > A, c.688G > A). In addition, a novel variant in ADGRA3 was identified (c.921-1G > A). CONCLUSIONS: This study reveals novel and known variants in PDE6A and PDE6B genes in Chinese families with autosomal recessive RP, and expands the clinical and genetic findings of photoreceptor-specific enzyme deficiencies.

Pde6brd1 mutation modifies cataractogenesis in Foxe3rct mice.

The Foxe3rct mutation, which causes early-onset cataracts, is a recessive mutation found in SJL/J mice. A previous study reported that cataract phenotypes are modified by the genetic background of mouse inbred strains and that the Pde6brd1 mutation, which induced degeneration of the photoreceptor cells, is a strong candidate genetic modifier to accelerate the severity of cataractogenesis of Foxe3rct mice. We created congenic mice by transferring a genomic region including the Foxe3rct mutation to the B6 genetic background, which does not carry the Pde6brd1 mutation. In the congenic mice, the cataract phenotypes became remarkably mild, and the development of cataracts was suppressed for a long time. Moreover, we created transgenic mice by injecting BAC clones including the wild-type Pde6b gene into the eggs of SJL-Foxe3rct mice. Although the resistant effect for cataract phenotypes in transgenic mice was less than that in congenic mice, the severity and onset time of cataract phenotypes were clearly improved and delayed, respectively, compared with the phenotypes of the original SJL-Foxe3rct mice. These results clearly show that the development of early-onset cataracts requires at least two mutant alleles of Foxe3rct and Pde6brd1, and another modifier associated with the severity of cataract phenotypes in Foxe3rct mice underlies the genetic backgrounds in mice.

Success of Gene Therapy in Late-Stage Treatment.

Retinal gene therapy has yet to achieve sustained rescue after disease onset - perhaps because transduction efficiency is insufficient ("too little") and/or the disease is too advanced ("too late") in humans. To test the latter hypothesis, we used a mouse model for retinitis pigmentosa (RP) that allowed us to restore the mutant gene in all diseased rod photoreceptor cells, thereby generating optimally treated retinas. We then treated mice at an advanced disease stage and analyzed the rescue. We showed stable, sustained rescue of photoreceptor structure and function for at least 1 year, demonstrating gene therapy efficacy after onset of degeneration. The results suggest that RP patients are treatable, even when the therapy is administered at late disease stages.

Next-generation sequencing targeted disease panel in rod-cone retinal dystrophies in Maori and Polynesian reveals novel changes and a common founder mutation.

IMPORTANCE: This study identifies unique genetic variation observed in a cohort of Maori and Polynesian patients with rod-cone retinal dystrophies using a targeted next-generation sequencing retinal disease gene panel. BACKGROUND: With over 250 retinal disease genes identified, genetic diagnosis is still only possible in 60-70% of individuals and even less within unique ethnic groups. DESIGN: Prospective genetic testing in patients with rod-cone retinal dystrophies identified from the New Zealand Inherited Retinal Disease Database, PARTICIPANTS: Sixteen patients of Maori and Polynesian ancestry. METHODS: Next-generation sequencing of a targeted retinal gene panel. Sanger sequencing for a novel PDE6B mutation in subsequent Maori patients. MAIN OUTCOME MEASURES: Genetic diagnosis, genotype-phenotype correlation. RESULTS: Thirteen unique pathogenic variants were identified in 9 of 16 (56.25%) patients in 10 different genes. A definitive genetic diagnosis was made in 7/16 patients (43.7%). Six changes were novel and not in public databases of human variation. In four patients, a homozygous, novel pathogenic variant (c.2197G > C, p.(Ala 733Pro)) in PDE6B was identified and also present in a further five similarly affected Maori patients. CONCLUSIONS AND RELEVANCE: Over half of the Maori and Polynesian patients with inherited rod-cone diseases have no pathogenic variant(s) detected with a targeted retinal next-generation sequencing strategy, which is supportive of novel genetic mechanisms in this population. A novel PDE6B founder variant is likely to account for 16% of recessive inherited retinal dystrophy in Maori. Careful characterization of the clinical presentation permits identification of further Maori patients with a similar phenotype and simplifies the diagnostic algorithm.

Cognitive decline, Aß pathology, and blood-brain barrier function in aged 5xFAD mice.

BACKGROUND: Patients with Alzheimer's disease (AD) develop blood-brain barrier dysfunction to varying degrees. How aging impacts Aß pathology, blood-brain barrier function, and cognitive decline in AD remains largely unknown. In this study, we used 5xFAD mice to investigate changes in Aß levels, barrier function, and cognitive decline over time. METHODS: 5xFAD and wild-type (WT) mice were aged between 9.5 and 15.5 months and tested for spatial learning and reference memory with the Morris Water Maze (MWM). After behavior testing, mice were implanted with acute cranial windows and intravenously injected with fluorescent-labeled dextrans to assess their in vivo distribution in the brain by two-photon microscopy. Images were processed and segmented to obtain intravascular intensity, extravascular intensity, and vessel diameters as a measure of barrier integrity. Mice were sacrificed after in vivo imaging to isolate brain and plasma for measuring Aß levels. The effect of age and genotype were evaluated for each assay using generalized or cumulative-linked logistic mixed-level modeling and model selection by Akaike Information Criterion (AICc). Pairwise comparisons were used to identify outcome differences between the two groups. RESULTS: 5xFAD mice displayed spatial memory deficits compared to age-matched WT mice in the MWM assay, which worsened with age. Memory impairment was evident in 5xFAD mice by 2-threefold higher escape latencies, twofold greater cumulative distances until they reach the platform, and twice as frequent use of repetitive search strategies in the pool when compared with age-matched WT mice. Presence of the rd1 allele worsened MWM performance in 5xFAD mice at all ages but did not alter the rate of learning or probe trial outcomes. 9.5-month-old 15.5-month-old 5xFAD mice had twofold higher brain Aß40 and Aß42 levels (p < 0.001) and 2.5-fold higher (p = 0.007) plasma Aß40 levels compared to 9.5-month-old 5xFAD mice. Image analysis showed that vessel diameters and intra- and extravascular dextran intensities were not significantly different in 9.5- and 15.5-month-old 5xFAD mice compared to age-matched WT mice. CONCLUSION: 5xFAD mice continue to develop spatial memory deficits and increased Aß brain levels while aging. Given in vivo MP imaging limitations, further investigation with smaller molecular weight markers combined with advanced imaging techniques would be needed to reliably assess subtle differences in barrier integrity in aged mice.

Proteomics identifies multiple retinitis pigmentosa associated proteins involved in retinal degeneration in a mouse model bearing a Pde6b mutation.

Retinitis pigmentosa (RP) is a progressive and degenerative retinal disease resulting in severe vision loss. RP have been extensively studied for pathogenetic mechanisms and treatments. Yet there is little information about alterations of RP associated proteins in phosphodiesterase 6 beta (Pde6b) mutated model. To explore the roles of RP causing proteins, we performed a label free quantitative mass spectrometry based proteomic analysis in rd10 mouse retinas. 3737 proteins were identified at the degenerative time points in rd10 mice. 222 and 289 differentially expressed proteins (DEPs) (fold change, FC > 2, p < 0.05) were detected at 5 and 8 weeks. Based on Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses, visual perception and phototransduction were severely affected. The downregulated DEPs were significantly enriched in cilium assembly and protein localization. 25 decreased DEPs causing autosomal recessive/dominant retinitis pigmentosa were visualized by heatmaps. Protein-protein interaction network represented 13 DEPs interacted directly with Pde6b protein. 25 DEPs causing RP were involved in phototransduction, visual perception, response to stimulus, protein localization and cilium assembly pathways. The significantly reduced expressions of DEPs were further validated by quantitative reverse transcription polymerase chain reaction (qPCR), Western blots (WB) and immunohistochemistry (IHC). This study revealed the molecular mechanisms underlying early and late stage of RP, as well as changes of RP-causing proteins.

In vivo base editing rescues photoreceptors in a mouse model of retinitis pigmentosa.

Retinitis pigmentosa (RP) is a group of retinal diseases that cause the progressive death of retinal photoreceptor cells and eventually blindness. Mutations in the ß-domain of the phosphodiesterase 6 (Pde6b) gene are the most identified causes of autosomal recessive RP. Clinically, there is no effective treatment so far that can stop the progression of RP and restore the vision. Here, we report a base editing approach in which adeno-associated virus (AAV)-mediated adenine base editor (ABE) delivering to postmitotic photoreceptors was conducted to correct the Pde6b mutation in a retinal degeneration 10 (rd10) mouse model of RP. Subretinal delivery of AAV8-ABE corrected Pde6b mutation with averaging up to 20.79% efficiency at the DNA level and 54.97% efficiency at the cDNA level without bystanders, restored PDE6B expression, preserved photoreceptors, and rescued visual function. RNA-seq revealed the preservation of genes associated with phototransduction and photoreceptor survival. Our data have demonstrated that base editing is a potential gene therapy that could provide durable protection against RP.

High amplitude pulses on the same charge condition efficiently elicit bipolar cell-mediated retinal ganglion cell responses in the degenerate retina.

Retinal pigmentosa (RP) patients lose vision due to the loss of photoreceptors. Retinal prostheses bypass the dead photoreceptors by electrically stimulating surviving retinal neurons, such as bipolar cells or retinal ganglion cells (RGCs). In previous studies, stimulus charge has been mainly optimized to maximize the RGC response to electrical stimulation. This study aimed to investigate the effect of amplitude and duration even under the same charge condition on eliciting RGC spikes in the wild-type and degenerate retinas. Wild-type (WT) Sprague-Dawley rats were used as the normal retinal model, and Pde6b knockout rats were used as a retinal degeneration (RD) model. Electrically-evoked RGC spikes were recorded from isolated rat retinas using an 8 × 8 multielectrode array. The same charge was maintained (10 or 20 nC), and electrical stimulation was applied to WT and RD retinas, adjusting the amplitude and duration of the 1st phase of biphasic pulses. In the pulse modulation of the 1st phase, high amplitude (short duration) pulses induced more RGC spikes than low amplitude (long duration) pulses. Both WT and RD retinas showed a significant reduction in the number of RGC spikes upon stimulation with lower amplitude (longer duration) pulses. In clinical trials where stimulus charges are delivered to the degenerate retina of blind patients, high amplitude (short duration) pulses would help elicit more RGC spikes.

Identification and Characterization of Retinitis Pigmentosa in a Novel Mouse Model Caused by PDE6B-T592I.

The cGMP-phosphodiesterase 6 beta subunit (PDE6B) is an essential component in the phototransduction pathway for light responses in photoreceptor cells. PDE6B gene mutations cause the death of rod photoreceptors, named as hereditary retinitis pigmentosa (RP) in humans and retinal degeneration (RD) in rodents. Here, we report a new RD model, identified from a phenotypic screen of N-ethyl-N-nitrosourea (ENU)-induced mutant mice, which displays retinal degeneration caused by a point mutation in the Pde6b gene that results in PDE6B-T592I mutant protein. The homozygous mutant mice show an extensive loss of rod photoreceptors at the age of 3 weeks; unexpectedly, the loss of rod photoreceptors can be partly rescued by dark rearing. Thus, this RD mutant model displays a light-dependent loss of rod photoreceptors. Both western blot and immunostaining results show very low level of mutant PDE6B-T592I protein in the retina. Structure modeling suggests that the T592I mutation probably affects the function and stability of PDE6B protein by changing intramolecular interactions. We further demonstrate that the expression of wild-type PDE6B delivered by subretinally injected adeno-associated virus (rAAV) prevents photoreceptor cell death in this RD model in vivo. The PDE6B-T592I mutant is, therefore, a valuable RD model for evaluating rAAV-mediated treatment and for investigating the molecular mechanism of light-dependent rod photoreceptor cell death that is related to impaired PDE6B function.

Prime Editing for the Installation and Correction of Mutations Causing Inherited Retinal Disease: A Brief Methodology.

Inherited retinal diseases (IRDs) encompass a large heterogeneous group of rare blinding disorders whose etiology originates from mutations in the 280 genes identified to date. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems represent a promising avenue for the treatment of IRDs, as exemplified by FDA clinical trial approval of EDIT-101 (AGN-151587), which removes a deep intronic variant in the CEP290 gene that causes Leber congenital amaurosis (LCA) type 10. Prime editing is a novel double-strand break (DSB) independent CRISPR/Cas system which has the potential to correct all 12 possible transition and transversion mutations in addition to small deletions and insertions. Here, as a proof-of-concept study, we describe a methodology using prime editing for the in vitro installation and correction of the classical Pde6brd10 c.1678C > T (p.Arg560Cys) mutation which causes autosomal recessive retinitis pigmentosa (RP) in mice.

Pseudodominant Inheritance of Retinitis Pigmentosa Due to Mutations in the Phosphodiesterase 6B Gene: A Case Report.

Mutations in the phosphodiesterase 6B (PDE6B) gene are a rare cause of autosomal recessive retinitis pigmentosa (arRP). We report on a non-consanguineous family with a pseudodominant inheritance of RP due to PDE6B mutations. We conducted a chart review of four members of a Puerto Rican family who underwent a comprehensive ophthalmic evaluation by at least one of the authors. The mutational screening was done using a genotyping microarray provided by Invitae Corporation, using next-generation sequencing (NGS) technology. Genomic DNA obtained from saliva samples is enriched for targeted regions using a hybridization-based protocol and sequenced using Illumina technology. A descriptive analysis was done. Patient 1A had a normal ophthalmic examination and a heterozygous pathogenic variant in the PDE6B gene c.1540del PLeu514Trpfs*61. Patients 1B, 2A, and 2B had mid-peripheral retinitis pigmentosa, concentric visual field ring scotomata in both eyes (OU), extinguished electroretinogram (ERG), and homozygous pathogenic variants in the PDE6B gene c.1540del PLeu514Trpfs*61. Even though mutations in the PDE6B gene usually lead to arRP, they may be inherited in a pseudodominant pattern in geographically isolated populations. Genotyping studies in patients with RP are warranted to classify inheritance mode correctly.

Development of a novel knockout model of retinitis pigmentosa using Pde6b-knockout Long-Evans rats.

Although rats with melanin-pigmentated retinal pigment epithelial (RPE) cells are physiologically more appropriate models for human eye research than their albino counterparts, reliable models from the former strain are not available to study retinal degeneration. Here, we describe the development of a novel Pde6b-knockout Long-Evans (LE Pde6b KO) rat model that recapitulates key features of human retinitis pigmentosa (RP). After the generation of the Pde6b-knockout Sprague-Dawley rats with the CRISPR-Cpf1 system, the LE rat was back-crossed over 5 generations to develop the pigmented LE Pde6b KO strain. Interestingly, LE Pde6b KO displayed well-developed bone-spicule pigmentation; a hallmark of fundus in patients with RP which cannot be observed in non-pigmented albino rats. Moreover, the rat model showed progressive thinning of the retina, which was evident by intravital imaging with optical coherence tomography. Histologically, significant atrophy was observed in the outer nuclear layer. Functionally, LE Pde6b KO presented a marked decrease of amplitude level during electroretinogram testing, demonstrating significant loss of visual function. Therefore, these findings suggest that the LE Pde6b KO model robustly recapitulates the hallmark phenotype of RP. We believe that the LE Pde6b KO model may be used effectively for preclinical translational research to further study retinal degeneration.

Exogenous PDE5 Expression Rescues Photoreceptors in RD1 Mice.

BACKGROUND: Catalytic hydrolysis of cyclic guanosine monophosphate (cGMP) by phosphodiesterase 6 (PDE6) is critical in phototransduction signalling in photoreceptors. Mutations in the genes encoding any of the three PDE6 subunits are associated with retinitis pigmentosa, the most common form of inherited retinal diseases. The RD1 mouse carries a naturally occurring nonsense mutation in the Pde6b gene. The RD1 mouse retina rapidly degenerates and fails to form rod photoreceptor outer segments due to the elevated cGMP level and subsequent excessive Ca2+ influx. In this study, we aim to test whether the PDE5 expression, a non-photoreceptor-specific member of the PDE superfamily, rescues photoreceptors in the RD1 retina. METHODS: Electroporation used the PDE5 expression plasmid to transfect neonatal RD1 mice. The mouse retina degeneration was assessed by retinal sections' stains with DAPI. The expression and localization of phototransduction proteins in photoreceptors were analysed by immunostaining. The expression of proteins in cultured cells was analysed by immunoblotting. RESULTS: The exogenous PDE5 expression, a non-photoreceptor-specific member of the PDE superfamily, prevents photoreceptor degeneration in RD1 mice. Unlike endogenous photoreceptor-specific PDE6 localised in the outer segments of photoreceptors, ectopically- expressed PDE5 was distributed in inner segments and synaptic terminals. PDE5 also promoted the development of the outer segments in RD1 mice. PDE5 co-expression with rhodopsin in cultured cells showed enhanced rhodopsin expression. CONCLUSION: Lowering the cGMP level in photoreceptors by PDE5 is sufficient to rescue photoreceptors in RD1 retinas. cGMP may also play a role in rhodopsin expression regulation in photoreceptors.

Retinal neovascularization induced by mutant Vldlr gene inhibited in an inherited retinitis pigmentosa mouse model: an in-vivo study.

AIM: To explore whether the retinal neovascularization (NV) in a genetic mutant mice model could be ameliorated in an inherited retinitis pigmentosa (RP) mouse, which would help to elucidate the possible mechanism and prevention of retinal NV diseases in clinic. METHODS: The Vldlr -/- mice, the genetic mutant mouse model of retinal NV caused by the homozygous mutation of Vldlr gene, with the rd1 mice, the inherited RP mouse caused by homozygous mutation of Pde6b gene were bred. Intercrossing of the above two mice led to the birth of the F1 hybrids, further inbreeding of which gave birth to the F2 offspring. The ocular genotypes and phenotypes of the mice from all generations were examined, with the F2 offspring grouped according to the genotypes. RESULTS: The rd1 mice exhibited the RP phenotype of outer retinal degeneration and loss of retinal function. The Vldlr -/- mice exhibited the phenotype of retinal NV obviously shown by the fundus fluorescein angiography. The F1 hydrides, with the heterozygote genotype, exhibited no phenotypes of RP or retinal NV. The F2 offspring with homozygous genotypes were grouped into four subgroups. They were the F2-I mice with the wild-type Pde6b and Vldlr genes (Pde6b+/+ -Vldlr+/+ ), which had normal ocular phenotypes; the F2-II mice with homozygous mutant Vldlr gene (Pde6b+/+ -Vldlr-/- ), which exhibited the retinal NV phenotype; the F2-III mice with homozygous mutant Pde6b gene (Pde6b-/- -Vldlr+/+ ), which exhibited the RP phenotype. Specifically, the F2-IV mice with homozygous mutant Vldlr and Pde6b gene (Pde6b-/- -Vldlr-/- ) showed only the RP phenotype, without the signs of retinal NV. CONCLUSION: The retinal NV can be inhibited by the RP phenotype, which implies the role of a hyperoxic state in treating retinal NV diseases.

Patient-Specific Retinal Organoids Recapitulate Disease Features of Late-Onset Retinitis Pigmentosa.

Although an increasing number of disease genes have been identified, the exact cellular mechanisms of retinitis pigmentosa (RP) remain largely unclear. Retinal organoids (ROs) derived from the induced pluripotent stem cells (iPSCs) of patients provide a potential but unvalidated platform for deciphering disease mechanisms and an advantageous tool for preclinical testing of new treatments. Notably, early-onset RP has been extensively recapitulated by patient-iPSC-derived ROs. However, it remains a challenge to model late-onset disease in a dish due to its chronicity, complexity, and instability. Here, we generated ROs from late-onset RP proband-derived iPSCs harboring a PDE6B mutation. Transcriptome analysis revealed a remarkably distinct gene expression profile in the patient ROs at differentiation day (D) 230. Changes in the expression genes regulating cGMP hydrolysis prompted the elevation of cGMP levels, which was verified by a cGMP enzyme-linked immunosorbent assay (ELISA) in patient ROs. Furthermore, significantly higher cGMP levels in patient ROs than in control ROs at D193 and D230 might lead to impaired formation of synaptic connections and the connecting cilium in photoreceptor cells. In this study, we established the first late-onset RP model with a consistent phenotype using an in vitro cell culture system and provided new insights into the PDE6B-related mechanism of RP.

Multiple genetic mutations implicate spectrum of phenotypes in Bardet-Biedl syndrome.

Bardet-Biedl syndrome (BBS) is a clinically and genetically heterogeneous ciliopathy with several clinical features including retinitis pigmentosa, obesity, kidney dysfunction, postaxial polydactyly, behavioral dysfunction and hypogonadism with wide spectrum of additional features. With multiple phenotypes and heterogeneous distribution, it is unlikely that BBS is caused by single gene defect. We have performed clinical and genetic diagnosis of two individuals from an Indian family with classical BBS symptoms. Whole exome sequencing identified homozygous missense mutation in BBS10 gene, hemizygous missense AR and homozygous missense PDE6B mutations in the proband and affected sibling with BBS. Identification of BBS10 mutation along with AR and PDE6B gene mutation will expand the genetic and phenotypic spectrum in individuals with BBS.