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Quantitative data of biological systems provide valuable baseline information for understanding pathology, experimental perturbations, and computational modeling. In mouse colon, calcitonin gene-related peptide (CGRP) is expressed by myenteric neurons with multiaxonal (Dogiel type II) morphology, characteristic of intrinsic primary afferent neurons (IPANs). Analogous neurons in other species and gut regions represent 5-35% of myenteric neurons. We aimed to quantify proportions of CGRP-immunopositive (CGRP+) myenteric neurons. Colchicine-treated wholemount preparations of proximal, mid, and distal colon were labeled for HuC/D, CGRP, nitric oxide synthase (NOS), and peripherin (Per). The pan-neuronal markers (Hu+/Per+) co-labeled 94% of neurons. Hu+/Per- neurons comprised ∼6%, but Hu-/Per+ cells were rare. Thus, quantification was based on Hu+ myenteric neurons (8576 total; 1225 ± 239 per animal, n = 7). CGRP+ cell bodies were significantly larger than the average of all Hu+ neurons (329 ± 13 vs. 261 ± 12 μm

Peripherin-2 (PRPH2) gene variants are a well-established cause of multiple inherited maculopathies including central areolar choroidal dystrophy (CACD) and pattern dystrophy. In this familial case study, we present a 63-year-old proband who presented with visual acuity of 20/63 right eye and 20/100 left eye with a complaint of lowered visual acuity in the left eye for unknown duration. Fundus examination presented with unifocal atrophic lesions bilaterally. Multi-modal imaging was obtained and genetic testing (My Retina Tracker; Blueprint Genetics) was performed. The proband was monoallelic for a novel missense mutation within the PRPH2 gene (Arg203Pro) not previously found in the literature or large databases (gnomAD, ClinVar, and HGMD). Subsequent examination of the proband's mother, older sister (65 years old), younger sister (53), and daughter (35) found the novel mutation to segregate with maculopathy ranging from speckled fundus autofluorescence with EZ disruption and RPE attenuation on spectral domain optical coherence tomography to large unifocal atrophic lesions throughout the macula bilaterally. The purpose of this case report is to add to the literature of PRPH2-associated disease by providing a comprehensive fundus examination of a family with autosomal dominant PRPH2-associated maculopathy diagnosed as central areolar choroidal dystrophy and pattern dystrophy.

This study tested whether combined hyperbaric oxygen (HBO) and allogenic adipose-derived mesenchymal stem cells (ADMSCs) would be superior to either one for improving the locomotor recovery in rat after acute traumatic spinal cord injury (TSCI) in rat. Adult-male Sprague–Dawley rats were equally categorized into group 1 (sham-operated control), group 2 (TSCI), group 3 (TSCI + HBO for 1.5 h/day for 14 consecutive days after TSCI), group 4 (TSCI + ADMSCs/1.2 × 106 cells by intravenous injection at 3 h and days 1/2 after TSCI), and group 5 (TSCI + HBO + ADMSCs), euthanized, and spinal cord tissue was harvested by day 49 after TSCI. The protein expressions of oxidative-stress (NOX-1/NOX-2), inflammatory-signaling (TLR-4/MyD88/IL-1β/TNF-α/substance-p), cell-stress signaling (PI3K/p-AKT/p-mTOR), and the voltage-gated sodium channel (Nav1.3/1.8/1.9) biomarkers were highest in group 2, lowest in group 1, and significantly lower in group 5 than in groups 3/4 (all P

This study investigated the hypothesis that probiotics enhanced the therapeutic effect of adipose-derived mesenchymal stem cells (ADMSCs) on alleviating neuropathic pain (NP) due to chronic constriction injury (CCI) mainly through regulating the microbiota in rats. SD rats (n = 50) were categorized into group 1 (sham-control), group 2 (NP), group 3 (NP + probiotics (i.e., 1.5 billion C.F.U./day/rat, orally 3 h after NP procedure, followed by QOD 30 times)), group 4 (NP + ADMSCs (3.0 × 105 cells) 3 h after CCI procedure, followed by QOD six times (i.e., seven times in total, i.e., mimic a clinical setting of drug use) and group 5 (NP + probiotics + ADMSCs (3.0 × 105 cells)) and euthanized by day 60 after NP induction. By day 28 after NP induction, flow-cytometric analysis showed circulating levels of early (AN-V+/PI−) and late (AN-V+/PI+) apoptotic, and three inflammatory (CD11b-c+, Ly6G+ and MPO+) cells were lowest in group 1 and significantly progressively reduced in groups 2 to 5 (all p < 0.0001). By days 7, 14, 21, 28, and 60 after CCI, the thresholds of thermal paw withdrawal latency (PWL) and mechanical paw withdrawal threshold (PWT) were highest in group 1 and significantly progressively increased in groups 2 to 5 (all p < 0.0001). Numbers of pain-connived cells (Nav1.8+/peripherin+, p-ERK+/peripherin+, p-p38+/peripherin+ and p-p38+/NF200+) and protein expressions of inflammatory (p-NF-κB, IL-1ß, TNF-α and MMP-9), apoptotic (cleaved-caspase-3, cleaved-PARP), oxidative-stress (NOX-1, NOX-2), DNA-damaged (γ-H2AX) and MAPK-family (p-P38, p-JNK, p-ERK1/2) biomarkers as well as the protein levels of Nav.1.3, Nav.1.8, and Nav.1.9 in L4-L5 in dorsal root ganglia displayed an opposite pattern of mechanical PWT among the groups (all p < 0.0001). In conclusion, combined probiotic and ADMSC therapy was superior to merely one for alleviating CCI-induced NP mainly through suppressing inflammation and oxidative stress.

Peripherin (PRPH), a type III intermediate filament, assembles with neurofilaments in neurons of the peripheral nervous system, including lower motor neurons (LMN). To evaluate the role of PRPH in LMN degeneration, we assessed PRPH and neurofilament light chain (NfL) in cerebrospinal fluid (CSF) and serum of 91 patients with motor neuron diseases (MND) and 69 controls. Overall, we found PRPH to be more concentrated in serum than in CSF. Serum PRPH resulted significantly increased in MND patients but it was unrelated to CSF‐NfL or survival in the amyotrophic lateral sclerosis (ALS) subset. PRPH might represent a marker of LMN involvement.

Mutations in thePRPH2gene encoding the photoreceptor-specific protein PRPH2 (also known as peripherin-2 or rds) cause a broad range of autosomal dominant retinal diseases. Most of these mutations affect the structure of the light-sensitive photoreceptor outer segment, which is composed of a stack of flattened “disc” membranes surrounded by the plasma membrane. The outer segment is renewed on a daily basis in a process whereby new discs are added at the outer segment base and old discs are shed at the outer segment tip. New discs are formed as serial membrane evaginations, which eventually enclose through a complex process of membrane remodeling (completely in rods and partially in cones). As disc enclosure proceeds, PRPH2 localizes to the rims of enclosed discs where it forms oligomers which fortify the highly curved membrane structure of these rims. In this study, we analyzed the outer segment phenotypes of mice of both sexes bearing a single copy of either the C150S or the Y141C PRPH2 mutation known to prevent or increase the degree of PRPH2 oligomerization, respectively. Strikingly, both mutations increased the number of newly forming, not-yet-enclosed discs, indicating that the precision of disc enclosure is regulated by PRPH2 oligomerization. Without tightly controlled enclosure, discs occasionally over-elongate and form large membranous “whorls” instead of disc stacks. These data show that the defects in outer segment structure arising from abnormal PRPH2 oligomerization are manifested at the stage of disc enclosure.SIGNIFICANCE STATEMENTThe light-sensitive photoreceptor outer segment contains a stack of flattened “disc” membranes that are surrounded, or “enclosed,” by the outer segment membrane. Disc enclosure is an adaptation increasing photoreceptor light sensitivity by facilitating the diffusion of the second messenger along the outer segment axes. However, the molecular mechanisms by which photoreceptor discs enclose within the outer segment membrane remain poorly understood. We now demonstrate that oligomers of the photoreceptor-specific protein peripherin-2, or PRPH2, play an active role in this process. We further propose that defects in disc enclosure because of abnormal PRPH2 oligomerization result in major structural abnormalities of the outer segment, ultimately leading to loss of visual function and cell degeneration in PRPH2 mutant models and human patients.

PurposeTo investigate the potential interaction between PRPF31 and PRPH2.MethodsHEK293T and human retinal pigment epithelial cells 19 (APRE-19) were used for our experiments. eGFP and mCherry reporter expression vectors were constructed for PRPF31 and PRPH2, respectively. Immunoblotting and co-immunoprecipitation (Co-IP) were used for gene expression validation and protein interaction. Immunofluorescence staining assay was used to test the co-localization analysis of PRPF31 and PRPH2.ResultsPRPF31-eGFP and PRPH2-mcherry were highly expressed in HEK293T and APRE-19 cells on fluorescence microscopy and western blot. Co-IP experiments showed that PRPF31 could be pulled down with an anti-PRPH2 antibody. There was co-localization between PRPF31 and PRPH2 in HEK293T, APRE-19 and mouse retina.ConclusionCo-IP and co-localization experiments suggest that PRPF31 interacted with PRPH2.

Trafficking of photoreceptor membrane proteins from their site of synthesis in the inner segment (IS) to the outer segment (OS) is critical for photoreceptor function and vision. Here we evaluate the role of syntaxin 3 (STX3), in trafficking of OS membrane proteins such as peripherin 2 (PRPH2) and rhodopsin. Photoreceptor-specific Stx3 knockouts [Stx3(f/f(iCre75)) and Stx3(f/f(CRX-Cre))] exhibited rapid, early-onset photoreceptor degeneration and functional decline characterized by structural defects in IS, OS, and synaptic terminals. Critically, in the absence of STX3, OS proteins such as PRPH2, the PRPH2 binding partner, rod outer segment membrane protein 1 (ROM1), and rhodopsin were mislocalized along the microtubules to the IS, cell body, and synaptic region. We find that the PRPH2 C-terminal domain interacts with STX3 as well as other photoreceptor SNAREs, and our findings indicate that STX3 is an essential part of the trafficking pathway for both disc (rhodopsin) and rim (PRPH2/ROM1) components of the OS.

Molecular variant interpretation lacks disease gene-specific cohorts for determining variant enrichment in disease versus healthy populations. To address the molecular etiology of retinal degeneration, specifically the PRPH2-related retinopathies, we reviewed genotype and phenotype information obtained from 187 eyeGENE® participants from 161 families. Clinical details were provided by referring clinicians participating in the eyeGENE® Network. The cohort was sequenced for variants in PRPH2. Variant complementary DNA clusters and cohort frequency were compared to variants in public databases to help us to determine pathogenicity by current American College of Medical Genetics and Genomics/Association for Molecular Pathology interpretation criteria. The most frequent variant was c.828+3A>T, which affected 28 families (17.4%), and 25 of 79 (31.64%) variants were novel. The majority of missense variants clustered in the D2 intracellular loop of the peripherin-2 protein, constituting a hotspot. Disease enrichment was noted for 23 (29.1%) of the variants. Hotspot and disease-enrichment evidence modified variant classification for 16.5% of variants. The missense allele p.Arg172Trp was associated with a younger age of onset. To the best of our knowledge, this is the largest patient cohort review of PRPH2-related retinopathy. Large disease gene-specific cohorts permit gene modeling for hotspot and disease-enrichment analysis, providing novel variant classification evidence, including for novel missense variants.

Diabetic Retinopathy is prevalent among patients with uncontrolled hyperglycemia resulting in vision loss. Despite numerous challenges to create a link among these conditions, the characterization of pathological neovascularization causing retinal damage due to the prognosis of early non-proliferative diabetic retinopathy to late proliferative diabetic retinopathy needs deep understanding. In this study, meta-analysis-based integration of gene expression datasets for the fibrovascular membrane of PDR and neural retina of NPDR were compared, to investigate the differentially expressed genes involved in retinal angiogenesis. Human samples with gene expression profiling of the same experiment type and platform with sufficient information for analysis were included in the study. The studies from cell lines and non-human studies, human samples that include serum, cornea, lens, and/or other ocular tissues or fluids, and studies that lack basic information for analysis were excluded. The microarray datasets available in the Gene Expression Omnibus database of the early and late stages in DR were screened to find common gene expression profiles. Using the INMEX bioinformatics tool, significantly upregulated and downregulated genes in the neural retina of Non-Proliferative Diabetic Retinopathy and fibrovascular membrane of Proliferative Diabetic Retinopathy were compared and studied by the combine effect size method. Using the STRING database PPI network, 50 upregulated and 50 downregulated genes were used to find the key candidate genes involved in retinal disease/degeneration in eye/retinal tissues. In the extensive gene expression meta-analysis performed using INMEX bioinformatics tool, overall, 7935 differentially expressed genes were identified and the respective heatmap was created by using the visualization tools of INVEX. STRING database PPI network identified Retinol Binding Protein 3, Neural Retina Leucine Zipper, S-Antigen Visual Arrestin, Peripherin 2, and Aryl Hydrocarbon Receptor Interacting Protein Like-1 to be the most highly ranked hub genes. The newly discovered potential genes related to retinal angiogenesis causing FVM formation in DR may provide insight into the cellular pathogenesis of NPDR to PDR.

Intermediate filaments are the most heterogeneous class among cytoskeletal elements. While some of them have been well-characterized, little is known about peripherin. Peripherin is a class III intermediate filament protein with a specific expression in the peripheral nervous system. Epigenetic modifications are involved in this cell-type-specific expression. Peripherin has important roles in neurite outgrowth and stability, axonal transport, and axonal myelination. Moreover, peripherin interacts with proteins involved in vesicular trafficking, signal transduction, DNA/RNA processing, protein folding, and mitochondrial metabolism, suggesting a role in all these processes. This review collects information regarding peripherin gene regulation, post-translational modifications, and functions and its involvement in the onset of a number of diseases.

Peripherin-2 (PRPH2) is one of the causative genes of inherited retinal dystrophy. While the gene is relatively common in Caucasians, reports from Asian ethnicities are limited. In the present study, we report 40 Japanese patients from 30 families with PRPH2-associated retinal dystrophy. We identified 17 distinct pathogenic or likely pathogenic variants using next-generation sequencing. Variants p.R142W and p.V200E were relatively common in the cohort. The age of onset was generally in the 40’s; however, some patients had earlier onset (age: 5 years). Visual acuity of the patients ranged from hand motion to 1.5 (Snellen equivalent 20/13). The patients showed variable phenotypes such as retinitis pigmentosa, cone-rod dystrophy, and macular dystrophy. Additionally, intrafamilial phenotypic variability was observed. Choroidal neovascularization was observed in three eyes of two patients with retinitis pigmentosa. The results demonstrate the genotypic and phenotypic variations of the disease in the Asian cohort.

Peripherin-2 (PRPH2) is a transmembrane glycoprotein crucial for the morphogenesis and stabilization of the photoreceptor outer segments. Variations inClinical manifestations and multimodal imaging were presented, as well as treatment history and six-year follow-up. In addition, genetic testing was performed to confirm the diagnosis.In this report, we present an extremely rare case of choroidal neovascularization (CNV) secondary to pattern dystrophy simulating fundus flavimaculatus (PDSFF). Multimodal imaging showed typical symmetric yellow flecks in posterior pole and choroidal neovascularization requiring timely treatment. A novel nonsense variant of c.552 C G; p.Y184X inWe described a novel

PURPOSE: Variants in the ABCA4 gene are causal for a variety of retinal dystrophy phenotypes, including Stargardt disease (STGD1). However, 15% of patients who present with symptoms compatible with STGD1/ABCA4 disease do not have identifiable causal ABCA4 variants. We hypothesized that a case–control collapsing analysis in ABCA4-negative patients with compatible symptoms would provide an objective measure to identify additional disease genes. METHODS: We performed a genome-wide enrichment analysis of “qualifying variants”—ultrarare variants predicted to impact protein function—in protein-coding genes in 79 unrelated cases and 9028 unrelated controls. RESULTS: Despite modest sample size, two known retinal dystrophy genes, PRPH2 and CRX, achieved study-wide significance (p < 1.33 × 10(−6)) under a dominant disease model, and eight additional known retinal dystrophy genes achieved nominal significance (p < 0.05). Across these ten genes, the excess of qualifying variants explained up to 36.8% of affected individuals. Furthermore, under a recessive model, the cone–rod dystrophy gene CERKL approached study-wide significance. CONCLUSION: Our results indicate that case–control collapsing analyses can efficiently identify pathogenic variants in genes in non-ABCA4 retinal dystrophies. The genome-wide collapsing analysis framework is an objective discovery method particularly suitable in settings with overlapping disease phenotypes.

CMYA5 is a candidate gene for schizophrenia because of the genetic association of variant rs10043986 (C > T) to this severe mental disorder. Studies of CMYA5 and its gene product, myospryn, in the brain and neuronal cells have not been previously reported. The SNP rs10043986 changes the 4,063rd amino acid from Pro to Leu, which is likely to alter protein function. To understand its potential role in the brain, we examined the neuronal expression of myospryn and its binding partner, desmin, an intermediate filament (IF) protein, and investigated how the two alleles of myospryn affect its binding to desmin. Myospryn and desmin are shown to be expressed in the brain and myospryn is shown to localize to the cytoplasm and nucleus of myoblast, neuroblastoma, and glioblastoma cell lines. Peripherin and vimentin, known brain IF proteins, have high protein similarity to desmin but were found not to interact with myospryn using yeast two-hybrid (Y2H). Using a quantitative Y2H assay and surface plasmon resonance, the T allele (Leu) of rs10043986 was found to have stronger binding to desmin than the C allele (Pro). Based on findings described in this report, we hypothesize that the interaction between myospryn to IF provides structural support and efficient rearrangement of the cytoskeleton network during early neuritogenesis.

Retinitis pigmentosa (RP) (OMIM: 268000) is a rare, heterogeneous group of inherited ocular disorders that results in a progressive retinal degeneration.1, 2 The PRPH2 gene ({"type":"entrez-nucleotide","attrs":{"text":"NM_000322.4","term_id":"194248075","term_text":"NM_000322.4"}}NM_000322.4) (OMIM: 179605), also known as RDS, AOFMD, AVMD, CACD2, DS, MDBS1, PRPH, rd2, RP7 and TSPAN22, is located on chromosome 6p21.1 with three exons spanning 26 395 bp length in human genome (GRCh38/hg38) that encodes a putative protein with 346 amino acids.3 The PRPH2 protein ({"type":"entrez-protein","attrs":{"text":"NP_000313.2","term_id":"118572596","term_text":"NP_000313.2"}}NP_000313.2) is a member of the transmembrane 4 superfamily, also known as the tetraspanin family. The majority of the members are cell‐surface proteins which were identified by the presence of four hydrophobic domains. The PRPH2 protein is a membrane‐associated glycoprotein, which is restricted to the area of photoreceptor outer segment discs.4 PRPH2 functions as an adhesion molecule by stabilization and compaction of outer segment discs. PRPH2 and ROM1 (OMIM: 180721) can be assembled into noncovalent tetramers (heterodimer) in vivo using disulphide bonds and higher order disulphide‐linked oligomers, thereby involving in photoreceptor disc morphogenesis.5 Mutations in the PRPH2 gene are involved with assorted blinding diseases of the retina, inducing degenerations in both central retinal and peripheral retinal.6, 7, 8 The relationships between the mutations in the PRPH2 gene and the resultant diseases in the patients are variable; making genotype/phenotype correlations different. PRPH2 mutation in RP patients and genotype/phenotype relationship have not been well described in the Chinese population.

Contains fulltext : 244059.pdf (Publisher’s version ) (Open Access) Mutations in PRPH2, encoding peripherin-2, are associated with the development of a wide variety of inherited retinal diseases (IRDs). To determine the causality of the many PRPH2 variants that have been discovered over the last decades, we surveyed all published PRPH2 variants up to July 2020, describing 720 index patients that in total carried 245 unique variants. In addition, we identified seven novel PRPH2 variants in eight additional index patients. The pathogenicity of all variants was determined using the ACMG guidelines. With this, 107 variants were classified as pathogenic, 92 as likely pathogenic, one as benign, and two as likely benign. The remaining 50 variants were classified as variants of uncertain significance. Interestingly, of the total 252 PRPH2 variants, more than half (n = 137) were missense variants. All variants were uploaded into the Leiden Open source Variation and ClinVar databases. Our study underscores the need for experimental assays for variants of unknown significance to improve pathogenicity classification, which would allow us to better understand genotype-phenotype correlations, and in the long-term, hopefully also support the development of therapeutic strategies for patients with PRPH2-associated IRD.

Purposes: To study the clinical and genetic background of a series of Italian patients affected by pattern dystrophy (PD).Methods: We reviewed patients with a clinical diagnosis of PD examined at the Eye Clinic in Florence from 2012 to 2019. We took into consideration patients with a standard ophthalmological examination, personal and familial ophthalmological history, fundus imaging, and molecular genetic analysis of genes PRPH2 and BEST1. We labelled patients with BEST1 and PRPH2 mutations as m-PD group (mutated) whereas patients with no mutations in these 2 genes as nm-PD group (non-mutated).Results: Seventy-seven PD patients were assessed (average age 59.7 ± 14.2, range 31-88 years). Fifty patients were placed in the nm-PD group and 27 in the m-PD. Pathogenic BEST1 and PRPH2 mutations were detected in 7% and 22% of PD patients, respectively. In total, we reported 1 BEST1 and 8 PRPH2 novel mutations. Ten patients were characterized by drusen in the nm-PD group whereas in no patients in the m-PD group drusen were detected at the fundus.Conclusions: An important proportion of patients affected by PD showed BEST1 or PRPH2 mutations. Patients affected by drusen represent a different sub-phenotype. Genetic examination is recommended for a correct clinical management.

Sander et al. have parsed the lipid composition of native-source photoreceptor disks and found large differences in fatty acid unsaturation and chain length between the center and rim regions. They selectively copurified membrane proteins and lipids from each region in SMALPs using nanobodies and antibodies., Photoreceptors rely on distinct membrane compartments to support their specialized function. Unlike protein localization, identification of critical differences in membrane content has not yet been expanded to lipids, due to the difficulty of isolating domain-specific samples. We have overcome this by using SMA to coimmunopurify membrane proteins and their native lipids from two regions of photoreceptor ROS disks. Each sample's copurified lipids were subjected to untargeted lipidomic and fatty acid analysis. Extensive differences between center (rhodopsin) and rim (ABCA4 and PRPH2/ROM1) samples included a lower PC to PE ratio and increased LC- and VLC-PUFAs in the center relative to the rim region, which was enriched in shorter, saturated FAs. The comparatively few differences between the two rim samples likely reflect specific protein–lipid interactions. High-resolution profiling of the ROS disk lipid composition gives new insights into how intricate membrane structure and protein activity are balanced within the ROS, and provides a model for future studies of other complex cellular structures.

Enterovirus-A71 (EV-A71) has been associated with severe neurological forms of hand, foot, and mouth disease (HFMD). EV-A71 infects motor neurons at neuromuscular junctions (NMJs) to invade the central nervous system (CNS). Here, we investigate the role of peripherin (PRPH) during EV-A71 infection, a type III intermediate neurofilament involved in neurodegenerative conditions. In mice infected with EV-A71, PRPH co-localizes with viral particles in the muscles at NMJs and in the spinal cord. In motor neuron-like and neuroblastoma cell lines, surface-expressed PRPH facilitates viral entry, while intracellular PRPH influences viral genome replication through interactions with structural and non-structural viral components. Importantly, PRPH does not play a role during infection with coxsackievirus A16, another causative agent of HFMD rarely associated with neurological complications, suggesting that EV-A71 ability to exploit PRPH represents a unique attribute for successful CNS invasion. Finally, we show that EV-A71 also exploits some of the many PRPH-interacting partners. Of these, small GTP-binding protein Rac1 represents a potential druggable host target to limit neuroinvasion of EV-A71.

More than 200 different mutations in peripherin-2 (PRPH2) are associated with multiple subtypes of inherited retinal diseases (IRDs), including retinitis pigmentosa and cone or macular diseases. Our goal was to understand how the poorly characterized PRPH2 mutation p.Pro210Arg (P210R) affects visual function and retinal structure as well as gain insight into the mechanism driving the clinical pathology.Eleven patients had clinical assessments including best-corrected visual acuity (BCVA), full field and multifocal electroretinography (ERG), static (spot size V) and kinetic perimetry (Octopus 900), and dark-adapted chromatic (DAC; Medmont; spot size V) perimetry. Images were acquired with the Optos ultra-wide field camera and spectral-domain optical coherence tomography (SD-OCT). Molecular characteristics of the P210R mutant protein were evaluated in vitro.Patients with the P210R mutation had BCVA (Snellen) ranging from 20/15 to 20/80. Perimetry showed a reduction in sensitivity, while ERG findings suggested that cone function was more impaired than rod function. Scotomas were identified corresponding to atrophic retinal lesions. Imaging revealed heterogeneous outer retinal changes such as hyperfluorescent flecks, hypo-autofluorescence (AF) regions of atrophy, and thinning of the photoreceptor layer on SD-OCT. In vitro findings suggested that P210R-Prph2 retains the ability to interact with binding partner Rom1 but abnormally accumulates in the endoplasmic reticulum (ER), suggesting the protein does not fold properly.Rod and cone sensitivities were decreased in subjects with the P210R mutation in PRPH2. There was scotomatous vision loss that occurred within the macula, likely due to atrophy that occurs after drusen have formed and have begun to resolve. This suggests that although rod and cone photoreceptors are dependent on PRPH2, preventing blindness in this specific subgroup of patients could involve therapeutics that impede the formation or lifecycle of drusen.

Background Retinitis pigmentosa (RP) is a rare, progressive, and hereditary disorder that leads to the progressive loss of vision and visual field, and in some cases blindness. The specific relationship between RP and glaucoma has been debated for decades. Methods In this study, we examined a Han RP family with concomitant angle-closure glaucoma (ACG), performed an inductive analysis of their clinical features and assistant results, and applied whole-exome sequencing (WES) technology for a molecular diagnosis. Results A novel transversion mutation (c.626 T > A) was identified in the peripherin-2 (PRPH2) gene in the proband, resulting in the substitution of Valine to aspartic acid in codon 209. A full ophthalmic examination showed that the proband with the c.626 T > A mutation had a typical RP manifestation, with close angles; however, the proband’s elder brother, who lacked the novel mutation, had a normal fundus and open angles. Conclusion Our results extend the genetic mutation spectrum of PRPH2 in RP, and provide evidence to support a genetic correlation between RP and ACG.

The purpose of this study was to develop a flexible, cost-efficient, next-generation sequencing (NGS) protocol for genetic testing. Long-range polymerase chain reaction (PCR) amplicons of up to 20 kb in size were designed to amplify entire genomic regions for a panel (n = 35) of inherited retinal disease (IRD)-associated loci. Amplicons were pooled and sequenced by NGS. The analysis was applied to 227 probands diagnosed with IRD: (A) 108 previously molecularly diagnosed, (B) 94 without previous genetic testing, and (C) 25 undiagnosed after whole-exome sequencing (WES). The method was validated with 100% sensitivity on cohort A. Long-range PCR-based sequencing revealed likely causative variant(s) in 51% and 24% of proband from cohorts B and C, respectively. Breakpoints of 3 copy number variants (CNVs) could be characterized. Long-range PCR libraries spike-in extended coverage of WES. Read phasing confirmed compound heterozygosity in 5 probands. The proposed sequencing protocol provided deep coverage of the entire gene, including intronic and promoter regions. Our method can be used (i) as a first-tier assay to reduce genetic testing costs, (ii) to elucidate missing heritability cases, (iii) to characterize breakpoints of CNVs at nucleotide resolution, (iv) to extend WES data to non-coding regions by spiking-in long-range PCR libraries, and (v) to help with phasing of candidate variants., International Journal of Molecular Sciences, 22 (4), ISSN:1422-0067

ESRETNET Study Group, The ERDC Study Group, The Associated Clinical Study Group., Inherited retinal diseases (IRDs), defined by dysfunction or progressive loss of photoreceptors, are disorders characterized by elevated heterogeneity, both at the clinical and genetic levels. Our main goal was to address the genetic landscape of IRD in the largest cohort of Spanish patients reported to date. A retrospective hospital-based cross-sectional study was carried out on 6089 IRD affected individuals (from 4403 unrelated families), referred for genetic testing from all the Spanish autonomous communities. Clinical, demographic and familiar data were collected from each patient, including family pedigree, age of appearance of visual symptoms, presence of any systemic findings and geographical origin. Genetic studies were performed to the 3951 families with available DNA using different molecular techniques. Overall, 53.2% (2100/3951) of the studied families were genetically characterized, and 1549 different likely causative variants in 142 genes were identified. The most common phenotype encountered is retinitis pigmentosa (RP) (55.6% of families, 2447/4403). The most recurrently mutated genes were PRPH2, ABCA4 and RS1 in autosomal dominant (AD), autosomal recessive (AR) and X-linked (XL) NON-RP cases, respectively; RHO, USH2A and RPGR in AD, AR and XL for non-syndromic RP; and USH2A and MYO7A in syndromic IRD. Pathogenic variants c.3386G > T (p.Arg1129Leu) in ABCA4 and c.2276G > T (p.Cys759Phe) in USH2A were the most frequent variants identified. Our study provides the general landscape for IRD in Spain, reporting the largest cohort ever presented. Our results have important implications for genetic diagnosis, counselling and new therapeutic strategies to both the Spanish population and other related populations., This work was supported by the Instituto de Salud Carlos III (ISCIII) of the Spanish Ministry of Health (FIS; PI16/00425 and PI19/00321), Centro de Investigación Biomédica en Red Enfermedades Raras (CIBERER, 06/07/0036), IIS-FJD BioBank (PT13/0010/0012), Comunidad de Madrid (CAM, RAREGenomics Project, B2017/BMD-3721), European Regional Development Fund (FEDER), the Organización Nacional de Ciegos Españoles (ONCE), Fundación Ramón Areces, Fundación Conchita Rábago and the University Chair UAM-IIS-FJD of Genomic Medicine. Irene Perea-Romero is supported by a PhD fellowship from the predoctoral Program from ISCIII (FI17/00192). Ionut F. Iancu is supported by a grant from the Comunidad de Madrid (CAM, PEJ-2017-AI/BMD7256). Marta del Pozo-Valero is supported by a PhD grant from the Fundación Conchita Rábago. Berta Almoguera is supported by a Juan Rodes program from ISCIII (JR17/00020). Pablo Minguez is supported by a Miguel Servet program from ISCIII (CP16/00116). Marta Corton is supported by a Miguel Servet program from ISCIII (CPII17/00006).

The large number of inherited retinal disease genes (IRD), including the photopigment rhodopsin and the photoreceptor outer segment (OS) structural component peripherin 2 (PRPH2), has prompted interest in identifying common cellular mechanisms involved in degeneration. Although metabolic dysregulation has been shown to play an important role in the progression of the disease etiology, identifying a common regulator that can preserve the metabolic ecosystem is needed for future development of neuroprotective treatments. Here, we investigated whether retbindin (RTBDN), a rod-specific protein with riboflavin binding capability, and a regulator of riboflavin-derived cofactors flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), is protective to the retina in different IRD models, one carrying the P23H mutation in rhodopsin (which causes retinitis pigmentosa) and one carrying the Y141C mutation in Prph2 (which causes a blended cone-rod dystrophy). RTBDN levels are significantly upregulated in both the rhodopsin (Rho)P23H/+ and Prph2Y141C/+ retinas. Rod and cone structural and functional degeneration worsened in models lacking RTBDN. In addition, removing Rtbdn worsened other phenotypes, such as fundus flecking. Retinal flavin levels were reduced in RhoP23H/+/Rtbdn&minus, /&minus, and Prph2Y141C/+/Rtbdn&minus, retinas. Overall, these findings suggest that RTBDN may play a protective role during retinal degenerations that occur at varying rates and due to varying disease mechanisms.

Objective: To determine the clinical relevance and frequency of BEST1 and PRPH2 mutations in a clinically diagnosed adult-onset vitelliform macular dystrophy (AVMD) group with Caucasian ethnicity. Methods: The study comprised 24 patients who had been diagnosed with AVMD via indirect fundus ophthalmoscopy and presented with a dome-shaped appearance between the retinal pigment epithelium and photoreceptors on their spectral-domain optical coherence tomography. They had lesion hyper- autofluorescence on their fundus autofluorescence images and were also investigated for BEST1 and PRPH2 mutations for a probable molecular aetiology. Results: No pathogenic or likely pathogenic mutation was detected in the BEST1 and PRPH2 genes of any of the clinically diagnosed AVDM patients. A heterozygous NM_000322.5:c.938C>T (p.Pro313Leu) variant of the PRPH2 gene was detected in 2 non-consanguineous patients. According to current guidelines, this variant was classified as a 'variant of uncertain significance'. Conclusion: In conclusion, AVMD is a genotypic and phenotypic heterogeneous disease. The genetic aetiology could not be explained by sequencing BEST1 and PRPH2 genes in the AVMD patients; however, the variant of PRPH2 could be a cause of predisposition relevant to the phenotype.

Genetic testing in a multisite clinical trial network for inherited eye conditions is described in this retrospective review of data collected through eyeGENE(®), the National Ophthalmic Disease Genotyping and Phenotyping Network. Participants in eyeGENE were enrolled through a network of clinical providers throughout the United States and Canada. Blood samples and clinical data were collected to establish a phenotype:genotype database, biorepository, and patient registry. Data and samples are available for research use, and participants are provided results of clinical genetic testing. eyeGENE utilized a unique, distributed clinical trial design to enroll 6,403 participants from 5,385 families diagnosed with over 30 different inherited eye conditions. The most common diagnoses given for participants were retinitis pigmentosa (RP), Stargardt disease, and choroideremia. Pathogenic variants were most frequently reported in ABCA4 (37%), USH2A (7%), RPGR (6%), CHM (5%), and PRPH2 (3%). Among the 5,552 participants with genetic testing, at least one pathogenic or likely pathogenic variant was observed in 3,448 participants (62.1%), and variants of uncertain significance in 1,712 participants (30.8%). Ten genes represent 68% of all pathogenic and likely pathogenic variants in eyeGENE. Cross-referencing current gene therapy clinical trials, over a thousand participants may be eligible, based on pathogenic variants in genes targeted by those therapies. This article is the first summary of genetic testing from thousands of participants tested through eyeGENE, including reports from 5,552 individuals. eyeGENE provides a launching point for inherited eye research, connects researchers with potential future study participants, and provides a valuable resource to the vision community.

Sympathetic neurons (SNs) capable of modulating the heart rate of murine cardiomyocytes (CMs) can be differentiated from human stem cells. The electrophysiological properties of human stem cell-derived SNs remain largely uncharacterized, and human neurocardiac cocultures remain to be established. Here, we have adapted previously published differentiation and coculture protocols to develop feeder-free SNs using human-induced pluripotent stem cells (hiPSCs). hiPSC-SNs were characterized in monoculture and coculture with hiPSC-CMs, using antibody labeling, enzyme-linked immunosorbent assay, and whole cell patch-clamp electrophysiology techniques. hiPSC-SNs stained positive for peripherin, tyrosine hydroxylase, and nicotinic acetylcholine receptors, the latter two colocalizing in somas and synaptic varicosities. hiPSC-SNs functionally matured in vitro and exhibited healthy resting membrane potentials (average = -61 ± 0.7 mV), secreted norepinephrine upon activation, and generated synaptic and action currents and inward and outward voltage-dependent currents. All hiPSC-SNs fired action potentials in response to current injection, local application of potassium, or spontaneously, followed by short-medium afterhyperpolarizations. hiPSC-SNs could successfully be maintained in coculture with hiPSC-CMs, and this induced further development of hiPSC-SN action potential kinetics. To test functional coupling between the neurons and cardiomyocytes, the hiPSC-CM beating response to nicotine-induced norepinephrine release was assessed. In neurocardiac cocultures, nicotine exposure significantly increased the hiPSC-CM spontaneous beating rate, but not in hiPSC-CM monocultures, supporting nicotinic neuronal hiPSC-SN stimulation directly influencing hiPSC-CM function. Our data show the development and characterization of electrophysiologically functional hiPSC-SNs capable of modulating the beating rate of hiPSC-CMs in vitro. These human cocultures provide a novel multicellular model to study neurocardiac modulation under physiological and pathological conditions.

Peripherin 2 (PRPH2) is a tetraspanin protein concentrated in the light-sensing cilium (called the outer segment) of the vertebrate photoreceptor. The mechanism underlying the ciliary targeting of PRPH2 and the etiology of cone dystrophy caused by PRPH2 mutations remain elusive. Here we show that the late endosome (LE) is the main waystation that critically sorts newly synthesized PRPH2 to the cilium. PRPH2 is expressed in the luminal membrane of the LE. We delineate multiple C-terminal motifs of PRPH2 that distinctively regulate its LE and ciliary targeting through ubiquitination and binding to ESCRT (Endosomal Sorting Complexes Required for Transport) component Hrs. Using the newly developed TetOn-inducible system in transfected male and female mouse cones in vivo, we show that the entry of nascent PRPH2 into the cone outer segment can be blocked by either cone dystrophy-causing C-terminal mutations of PRPH2, or by short-term perturbation of the LE or recycling endosomal traffic. These findings open new avenues of research to explore the biological role of the LE in the biosynthetic pathway and the etiology of cone dystrophy caused by PRPH2 mutations and/or malfunctions of the LE. SIGNIFICANCE STATEMENT Peripherin 2 (PRPH2) is a tetraspanin protein abundantly expressed in the light-sensing cilium, the outer segment, of the vertebrate photoreceptor. The mechanism underlying the ciliary transport of PRPH2 is unclear. The present study reveals a novel ciliary targeting pathway, in which the newly synthesized PRPH2 is first targeted to the lumen of the late endosome (LE) en route to the cilia. We deciphered the protein motifs and the machinery that regulates the LE trafficking of PRPH2. Using a novel TetOn-inducible system in transfected mouse cones, we showed that the LE pathway of PRPH2 is critical for its outer segment expression. A cone dystrophy-causing mutation impairs the LE and ciliary targeting of PRPH2, implicating the relevance of LE to cone/macular degenerative diseases.

Peripherin 2 (PRPH2) is a retina-specific tetraspanin protein essential for the formation of rod and cone photoreceptor outer segments (OS). Patients with mutations in PRPH2 exhibit severe retinal degeneration characterized by vast inter- and intra-familial phenotypic heterogeneity. To help understand contributors to this within-mutation disease variability, we asked whether the PRPH2 binding partner rod OS membrane protein 1 (ROM1) could serve as a phenotypic modifier. We utilized knockin and transgenic mouse models to evaluate the structural, functional and biochemical effects of eliminating one allele of Rom1 (Rom1+/−) in three different Prph2 models which mimic human disease: C213Y Prph2 (Prph2C/+), K153Del Prph2 (Prph2K/+) and R172W (Prph2R172W). Reducing Rom1 in the absence of Prph2 mutations (Rom1+/−) had no effect on retinal structure or function. However, the effects of reducing Rom1 in the presence of Prph2 mutations were highly variable. Prph2K/+/Rom1+/− mice had improved rod and cone function compared with Prph2K/+ as well as amelioration of K153Del-associated defects in PRPH2/ROM1 oligomerization. In contrast, Prph2R172W/Rom1+/− animals had worsened rod and cone function and exacerbated retinal degeneration compared with Prph2R172W animals. Removing one allele of Rom1 had no effect in Prph2C/+. Combined, our findings support a role for non-pathogenic ROM1 null variants in contributing to phenotypic variability in mutant PRPH2-associated retinal degeneration. Since the effects of Rom1 reduction are variable, our data suggest that this contribution is specific to the type of Prph2 mutation.

Purpose Age-related macular degeneration (AMD) is associated with multiple environmental and genetic risk factors. Two main risk factors for AMD are variants in the CFH and ARMS2/HTRA1 genes. We investigated over 2000 variants in AMD patients and controls using high-throughput sequencing methods to search for variants associated with AMD. Methods A total of 296 AMD patients and 100 controls were enrolled in this study. Genetic analysis was performed with the Illumina NextSeq 500 system. Results Multivariate analysis of patients and controls, adjusted for age, sex and smoking status (pack-years), revealed that three SNPs were strong risk factors independently associated with AMD: CFH Y402H, ARMS A69S and PRPH2 c.582-67T>A (rs3818086). The TC genotype in CFH Y402H was associated with 1.90-fold higher odds, and the CC genotype was associated with 5.66-fold higher odds of AMD compared with the TT genotype. The GT genotype in ARMS A69S was associated with 2.40-fold higher odds, and the TT genotype was associated with 6.75-fold higher odds of disease compared with the GG genotype. In the case of rs3818086, the A allele could be considered a 'risk' allele, since AA + TA genotypes were associated with 2.33-fold higher odds of AMD compared with the TT genotype. Conclusions Although PRPH2 mutations have been previously implicated in various forms of retinal degeneration, to the best of our knowledge, this study is the first to show that the rs3818086 variant increases the risk for AMD more than two times. Further studies on larger cohorts are required to elucidate how this variant affects protein structure.

Purpose Heterozygous peripherin mutation is associated with a wide range of typically adult-onset retinal phenotypes which can include asymptomatic maculopathy. There are few reports of biallelic peripherin mutations, only one of which detailed the ophthalmic phenotype. This report documents the retinal phenotype associated with homozygosity for a known peripherin mutation (c.659G>A; p.Arg220Gln), highlights its similar appearance to what was described in the one previous report, and shows how examination of family members can be useful in genetic diagnosis. Methods Retrospective case series. Results A 13-year-old Emirati boy was referred for low vision. The parents felt he was blind at birth but noted improvement with time. Retinal examination was significant for central macula horizontal ovoid discoloration as was documented for young adults with homozygous peripherin mutations in the one previous report. Electroretinography revealed cone-rod dysfunction. Both asymptomatic parents were examined and found to have central macular abnormalities. Sanger sequencing of peripherin based on clinical features uncovered the pathogenic variant c.659G>A; p.Arg22Gln (NM_000322.4) in homozygosity in the child and in heterozygosity in each parent. Exome sequencing in the child excluded pathologic variants in other retinal dystrophy genes. Conclusions The experience with this family highlights clinical features suggestive for biallelic peripherin mutations, documents cone-rod dysfunction as associated with homozygosity for the p.Arg220Gln peripherin mutation, and is an example of how examination of family members can help to guide genetic testing.