Publications by authors named "Vinit B Mahajan"

181 Publications

Genetics of Uveitis.

Ocul Immunol Inflamm 2021 Feb;29(2):215-218

Centre for Ophthalmology, University Tuebingen, Tuebingen, Germany.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/09273948.2021.1910430DOI Listing
February 2021

Structure-based phylogeny identifies Avoralstat as a TMPRSS2 inhibitor that prevents SARS-CoV-2 infection in mice.

J Clin Invest 2021 Apr 12. Epub 2021 Apr 12.

Department of Ophthalmology, Stanford University, Palo Alto, United States of America.

Drugs targeting host proteins can act prophylactically to reduce viral burden early in disease and limit morbidity, even with antivirals and vaccination. Transmembrane serine protease 2 (TMPRSS2) is a human protease required for SARS-CoV-2 viral entry and may represent such a target. We hypothesized that drugs selected from proteins related by their tertiary structure, rather than their primary structure, were likely to interact with TMPRSS2. We created a structure-based phylogenetic computational tool named 3DPhyloFold to systematically identify structurally similar serine proteases with known therapeutic inhibitors and demonstrated effective inhibition of SARS-CoV-2 infection in vitro and in vivo. Several candidate compounds, Avoralstat, PCI-27483, Antipain, and Soybean-Trypsin-Inhibitor, inhibited TMPRSS2 in biochemical and cell infection assays. Avoralstat, a clinically tested Kallikrein-related B1 inhibitor, inhibited SARS-CoV-2 entry and replication in human airway epithelial cells. In an in vivo proof of principle, Avoralstat significantly reduced lung tissue titers and mitigated weight-loss when administered prophylactically to SARS-CoV-2 susceptible mice indicating its potential to be repositioned for COVID-19 prophylaxis in humans.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1172/JCI147973DOI Listing
April 2021

Telegenetics for inherited retinal diseases in the COVID-19 environment.

Int J Retina Vitreous 2021 Mar 29;7(1):25. Epub 2021 Mar 29.

Molecular Surgery Laboratory, Department of Ophthalmology, Byers Eye Institute, Stanford University, Palo Alto, CA, 94304, USA.

Inherited retinal diseases (IRDs) are visually debilitating conditions that affect families worldwide. They require extensive clinical testing, examination, and patient and family counseling, which are frequently accomplished over single-day extended clinic visits. However, the COVID-19 pandemic has limited the number of patients and staff allowed in clinics, leading to interruptions in care. We therefore developed telehealth management protocols for complete or hybrid virtual visits. The three main components of our telegenetics approach included reviewing the diagnostic tests results remotely, in-person or virtual video visits with a retina specialist, and virtual genetic testing using saliva kits. During the first 5 months of the program, telegenetic care was provided for 80 patients, including 3 international patients, and a spectrum of retinal dystrophies were diagnosed and managed. In conclusion, telegenetic virtual visits ensure continuity of care while reducing patient and provider exposure to SARS-CoV-2 and may continue and expand into other medical genetic conditions long after the pandemic.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s40942-021-00301-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8006125PMC
March 2021

Peptidomimetics Therapeutics for Retinal Disease.

Biomolecules 2021 Feb 24;11(3). Epub 2021 Feb 24.

Molecular Surgery Laboratory, Department of Ophthalmology, Byers Eye Institute, Stanford University, Palo Alto, CA 94304, USA.

Ocular disorders originating in the retina can result in a partial or total loss of vision, making drug delivery to the retina of vital importance. However, effectively delivering drugs to the retina remains a challenge for ophthalmologists due to various anatomical and physicochemical barriers in the eye. This review introduces diverse administration routes and the accordant pharmacokinetic profiles of ocular drugs to aid in the development of safe and efficient drug delivery systems to the retina with a focus on peptidomimetics as a growing class of retinal drugs, which have great therapeutic potential and a high degree of specificity. We also discuss the pharmacokinetic profiles of small molecule drugs due to their structural similarity to small peptidomimetics. Lastly, various formulation strategies are suggested to overcome pharmacokinetic hurdles such as solubility, retention time, enzymatic degradation, tissue targeting, and membrane permeability. This knowledge can be used to help design ocular delivery platforms for peptidomimetics, not only for the treatment of various retinal diseases, but also for the selection of potential peptidomimetic drug targets.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/biom11030339DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7995992PMC
February 2021

Molecular Surgery: Proteomics of a Rare Genetic Disease Gives Insight into Common Causes of Blindness.

iScience 2020 Nov 13;23(11):101667. Epub 2020 Oct 13.

Omics Laboratory, Stanford University, Palo Alto, CA, USA.

Rare diseases are an emerging global health priority. Although individually rare, the prevalence of rare "orphan" diseases is high, affecting approximately 300 million people worldwide. Treatments for these conditions are often inadequate, leaving the disease to progress unabated. Here, we review the clinical features and pathophysiology of neovascular inflammatory vitreoretinopathy (NIV), a rare inflammatory retinal disease caused by mutations in the gene. Although the prevalence of NIV is low (1 in 1,000,000 people), the disease mimics more common causes of blindness (e.g. uveitis, retinitis pigmentosa, proliferative diabetic retinopathy, and proliferative vitreoretinopathy) at distinct clinical stages. There is no cure for NIV to date. We highlight how personalized proteomics helped identify potential stage-specific biomarkers and drug targets in liquid vitreous biopsies. The NIV vitreous proteome revealed enrichment of molecular pathways associated with common retinal pathologies and implicated superior targets for therapeutic drug repositioning. In addition, we review our pipeline for collecting, storing, and analyzing ophthalmic surgical samples. This approach can be adapted to treat a variety of rare genetic diseases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.isci.2020.101667DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7586135PMC
November 2020

Retinal Manifestations of Mitochondrial Oxidative Phosphorylation Disorders.

Invest Ophthalmol Vis Sci 2020 10;61(12):12

Jonas Children's Vision Care, Department of Ophthalmology, Columbia University Irving Medical Center, New York, New York, United States.

Purpose: The purpose of this paper was to discuss manifestations of primary mitochondrial dysfunctions and whether the retinal pigment epithelium or the photoreceptors are preferentially affected.

Methods: A retrospective analysis was performed of patients with clinically and laboratory confirmed diagnoses of maternally inherited diabetes and deafness (MIDD) or Kearns-Sayre syndrome (KSS). Patients underwent full ophthalmic examination, full-field electroretinogram, and multimodal imaging studies, including short-wavelength autofluorescence, spectral domain-optical coherence tomography, and color fundus photography.

Results: A total of five patients with MIDD and four patients with KSS were evaluated at two tertiary referral centers. Mean age at initial evaluation was 50.3 years old. Nascent outer retinal tubulations corresponding with faint foci of autofluorescence were observed in two patients with MIDD. Characteristic features of this cohort included a foveal sparing phenotype observed in 13 of 18 eyes (72%), global absence of intraretinal pigment migration, and preserved retinal function on full-field electroretinogram testing in 12 of 16 eyes (75%). One patient diagnosed with MIDD presented with an unusual pattern of atrophy surrounding the parapapillary region and one patient with KSS presented with an atypical choroideremia-like phenotype.

Conclusions: MIDD and KSS are phenotypically heterogeneous disorders. Several features of disease suggest that primary mitochondrial dysfunction may first affect the retinal pigment epithelium followed by secondary photoreceptor loss. Similarities between primary mitochondrial degenerations and retinal disorders, such as age-related macular degeneration may suggest a primary role of mitochondria in the pathogenesis of these oligogenic disorders.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1167/iovs.61.12.12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7571321PMC
October 2020

Sex Does Not Influence Visual Outcomes After Blast-Mediated Traumatic Brain Injury but IL-1 Pathway Mutations Confer Partial Rescue.

Invest Ophthalmol Vis Sci 2020 10;61(12)

Department of Pediatrics, University of Iowa, Iowa City, Iowa, United States.

Purpose: In a mouse model of blast-mediated traumatic brain injury (bTBI), interleukin-1 (IL-1)-pathway components were tested as potential therapeutic targets for bTBI-mediated retinal ganglion cell (RGC) dysfunction. Sex was also evaluated as a variable for RGC outcomes post-bTBI.

Methods: Male and female mice with null mutations in genes encoding IL-1α, IL-1β, or IL-1RI were compared to C57BL/6J wild-type (WT) mice after exposure to three 20-psi blast waves given at an interblast interval of 1 hour or to mice receiving sham injury. To determine if genetic blockade of IL-1α, IL-1β, or IL-1RI could prevent damage to RGCs, the function and structure of these cells were evaluated by pattern electroretinogram and optical coherence tomography, respectively, 5 weeks following blast or sham exposure. RGC survival was also quantitatively assessed via immunohistochemical staining of BRN3A at the completion of the study.

Results: Our results showed that male and female WT mice had a similar response to blast-induced retinal injury. Generally, constitutive deletion of IL-1α, IL-1β, or IL-1RI did not provide full protection from the effects of bTBI on visual outcomes; however, injured WT mice had significantly worse visual outcomes compared to the injured genetic knockout mice.

Conclusions: Sex does not affect RGC outcomes after bTBI. The genetic studies suggest that deletion of these IL-1 pathway components confers some protection, but global deletion from birth did not result in a complete rescue.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1167/iovs.61.12.7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7582458PMC
October 2020

Intravitreal methotrexate and fluocinolone acetonide implantation for Vogt-Koyanagi-Harada uveitis.

Am J Ophthalmol Case Rep 2020 Sep 2;19:100859. Epub 2020 Aug 2.

Department of Ophthalmology, Byers Eye Institute, Stanford University, Palo Alto, CA, USA.

Purpose: To report a case of intravitreal methotrexate treatment and fluocinolone acetonide (Retisert®) implantation in a patient with Vogt-Koyanagi-Harada syndrome (VKH).

Observations: A 34-year-old male was referred for worsening vision and bilateral panuveitis consistent with VKH. He was treated with prednisone, mycophenolate mofetil, prednisolone acetate eye drops, and injections of triamcinolone and adalimumab. He failed to improve with these therapies and developed multiple adverse effects, including hepatotoxicity, severe eye pain, cataracts, and cystoid macular edema. We treated him with intravitreal methotrexate injections in both eyes, which rapidly improved his eye pain, inflammation, and vision. He subsequently underwent fluocinolone acetonide (Retisert®) implantation, cataract extraction with intraocular lens insertion, and Ahmed tube placement for long-term intraocular pressure control. His vision improved from hand motions to 20/30, intraocular pressure remained stable at 17, there was complete resolution of his panuveitis and uveitic macular edema, and his systemic medications were able to be discontinued.

Conclusions: /Importance: This case demonstrates intravitreal methotrexate may successfully treat intraocular inflammation, pain, and macular edema in VKH. Excellent long-term vision and reduction of adverse effects of systemic medications were also achieved with subsequent fluocinolone acetonide implantation. Combining these two targeted therapies may be an effective strategy in treating VKH in patients who have severe pain and cannot tolerate systemic therapy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ajoc.2020.100859DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7415829PMC
September 2020

Whole-Exome Sequencing of Patients With Posterior Segment Uveitis.

Am J Ophthalmol 2021 01 21;221:246-259. Epub 2020 Jul 21.

Molecular Surgery Laboratory, Stanford University, Palo Alto, California, USA; Department of Ophthalmology, Byers Eye Institute, Stanford University, Palo Alto, California, USA; Veterans Affairs, Palo Alto HCS, Palo Alto, California, USA. Electronic address:

Objective: To elucidate molecular risk factors for posterior segment uveitis using a functional genomics approach.

Design: Genetic association cohort study.

Methods: Setting: Single-center study at an academic referral center.

Study Population: 164 patients with clinically diagnosed uveitis of the posterior segment.

Main Outcome Measures: Exome sequencing was used to detect variants identified in 164 patients with posterior segment uveitis. A phenotype-driven analysis, protein structural modeling, and in silico calculations were then used to rank and predict the functional consequences of key variants.

Results: A total of 203 single nucleotide variants, in 23 genes across 164 patients, were included in this study. Both known and novel variants were identified in genes previously implicated in specific types of syndromic uveitis-such as NOD2 (Blau syndrome) and CAPN5 NIV (neovascular inflammatory vitreoretinopathy)-as well as variants in genes not previously linked to posterior segment uveitis. Based on a ranked list and protein-protein-interaction network, missense variants in NOD-like receptor family genes (NOD2, NLRC4, NLRP3, and NLRP1), CAPN5, and TYK2 were characterized via structural modeling and in silico calculations to predict how specific variants might alter protein structure and function. The majority of analyzed variants were notably different from wild type.

Conclusions: This study implicates new pathways and immune signaling proteins that may be associated with posterior segment uveitis susceptibility. A larger cohort and functional studies will help validate the pathogenicity of the mutations identified. In specific cases, whole-exome sequencing can help diagnose nonsyndromic uveitis in patients harboring known variants for syndromic inflammatory diseases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ajo.2020.07.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7736069PMC
January 2021

Sex Differences in the Repair of Retinal Detachments in the United States.

Am J Ophthalmol 2020 11 5;219:284-294. Epub 2020 Jul 5.

Byers Eye Institute at the Stanford University School of Medicine, Palo Alto, California, USA.

Purpose: To investigate differences between women and men in the repair of rhegmatogenous retinal detachments (RRDs) in the United States.

Design: Retrospective cohort study.

Methods: Setting: A large insurance claims database.

Participants: Subjects with an incident RRD between 2007 and 2015.

Data: Demographic data, comorbid ocular conditions associated with RRD, systemic comorbidities, and surgical intervention (pneumatic retinopexy [PR], pars plana vitrectomy [PPV], laser barricade, or scleral buckle [SB]) were collected.

Main Outcome Measures: Odds of receipt of surgical intervention for incident RRD, time to repair, type of intervention, and the rate of reoperation by sex.

Results: The study period included 133 million eligible records with 61,071 cases of incident RRD among which 43% (n = 26,289) were women. The primary outcome model had 23,933 confirmed RRD cases with a 93% retinal detachment repair rate. Women had 34% reduced odds of receipt of surgical repair of an RRD (odds ratio [OR] 0.66, 95% confidence interval [CI] 0.59, 0.73; P < .001) after adjusting for confounders. This effect persisted in all sensitivity models. Among patients who received repair, women were more often delayed (0.17 days, P = .04). Women were more likely to undergo primary laser barricade (relative risk ratio [RRR] 1.68, P < .001), primary SB (RRR 1.15, P < .001), and PR (RRR 1.07, P < .04) than men. The odds of reoperation were lower in women (OR 0.91, 95% CI 0.85, 0.96; P = .002) after adjustment.

Conclusions: Insured women are less likely than insured men to receive surgical intervention for an RRD. Based on the results of this study, if the odds of repair were equal between women and men in the United States, then 781 more women would receive surgery each year, or 7,029 more during the study period. Women are more likely to have the repair performed with scleral buckle, laser barricade, and pneumatic retinopexy. The reason for these sex differences in RRD repair remains unknown and requires further investigation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ajo.2020.06.039DOI Listing
November 2020

Quantitative Autofluorescence Following Gene Therapy With Voretigene Neparvovec.

JAMA Ophthalmol 2020 08;138(8):919-921

Department of Ophthalmology, Columbia University Irving Medical Center, New York, New York.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1001/jamaophthalmol.2020.2018DOI Listing
August 2020

Optical Gap Biomarker in Cone-Dominant Retinal Dystrophy.

Am J Ophthalmol 2020 10 21;218:40-53. Epub 2020 May 21.

Jonas Children's Vision Care, Department of Ophthalmology, Columbia University Irving Medical Center, New York, New York, USA; Department of Pathology and Cell Biology, Columbia University Irving Medical Center, New York, New York, USA. Electronic address:

Purpose: To characterize the progression of optical gaps and expand the known etiologies of this phenotype.

Design: Retrospective cohort study.

Methods: Thirty-six patients were selected based on the identification of an optical gap on spectral-domain optical coherence tomography (OCT) from a large cohort of patients (N = 746) with confirmed diagnoses of inherited retinal dystrophy. The width and height of the gaps in 70 eyes of 36 patients were measured by 2 independent graders using the caliper tool on Heidelberg Explorer. Measurements of outer and central retinal thickness were also evaluated and correlated with gap dimensions.

Results: Longitudinal analysis confirmed the progressive nature of optical gaps in patients with Stargardt disease, achromatopsia, occult macular dystrophy, and cone dystrophies (P < .003). Larger changes in gap width were noted in patients with Stargardt disease (78.1 μm/year) and cone dystrophies (31.9 μm/year) compared with patients with achromatopsia (16.2 μm/year) and occult macular dystrophy (15.4 μm/year). Gap height decreased in patients with Stargardt disease (6.5 μm/year; P = .02) but increased in patients with achromatopsia (3.3 μm/year) and occult macular dystrophy (1.2 μm/year). Gap height correlated with measurements of central retinal thickness at the fovea (r = 0.782, P = .00012). Interocular discordance of the gap was observed in 7 patients. Finally, a review of all currently described etiologies of optical gap was summarized.

Conclusion: The optical gap is a progressive phenotype seen in an increasing number of etiologies. This progressive nature suggests a use as a biomarker in the understanding of disease progression. Interocular discordance of the phenotype may be a feature of Stargardt disease and cone dystrophies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ajo.2020.05.016DOI Listing
October 2020

Reply.

Retina 2020 08;40(8):e37

Department of Ophthalmology, Byers Eye Institute, Stanford University, Palo Alto, California.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1097/IAE.0000000000002828DOI Listing
August 2020

Proteomic analysis of intermediate uveitis suggests myeloid cell recruitment and implicates IL-23 as a therapeutic target.

Am J Ophthalmol Case Rep 2020 Jun 6;18:100646. Epub 2020 Mar 6.

Omics Laboratory, Stanford University, Palo Alto, CA, USA.

Purpose: To profile vitreous protein expression of intermediate uveitis (IU) patients.

Observations: We identified a mean of 363 ± 41 unique proteins (mean ± SD) in IU vitreous and 393 ± 69 unique proteins in control samples using liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of liquid vitreous biopsies collected during pars plana vitrectomy. A total of 233 proteins were differentially expressed among control and IU samples, suggesting a protein signature that could distinguish the two groups. Pathway analysis identified 22 inflammatory mediators of the interleukin-12 (IL-12) signaling pathway in IU vitreous. Upstream regulator analysis identified downstream mediators of IL-23 and myeloid differentiation primary response protein (MYD88), both of which are involved in the recruitment and differentiation of myeloid cells. Taken together, our results suggest the recruitment of myeloid cells as an upstream pathway in the pathogenesis of IU.

Conclusions: This study provides insights into proteins that will serve as biomarkers and therapeutic targets for IU. These biomarkers will help design future clinical trials using rational molecular therapeutics.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ajoc.2020.100646DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7132169PMC
June 2020

Phenotypic variance in Calpain-5 retinal degeneration.

Am J Ophthalmol Case Rep 2020 Jun 24;18:100627. Epub 2020 Feb 24.

Omics Laboratory, Byers Eye Institute, Department of Ophthalmology, Stanford University, Palo Alto, CA, USA.

Purpose: To characterize the phenotype of patients with mild calpain-5 Neovascular Inflammatory Vitreoretinopathy (ADNIV).

Observations: The p.R243L mutation is typically associated with onset in the twenties and severe, progressive uveitis, retinal neovascularization, and intraocular fibrosis. Two subjects with this variant only showed mild peripheral retinal pigmentary degeneration and loss of the ERG b-wave at age 45 and 69, respectively, without signs of uveitis or neovascularization.

Conclusions/importance: The phenotypic penetrance of a specific variant in CAPN5-vitreoretinopathy may vary significantly in severity. Patients with pigmentary retinal dystrophy may consider gene testing.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ajoc.2020.100627DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7132063PMC
June 2020

Modulation of Post-Traumatic Immune Response Using the IL-1 Receptor Antagonist Anakinra for Improved Visual Outcomes.

J Neurotrauma 2020 06 16;37(12):1463-1480. Epub 2020 Mar 16.

Department of Pediatrics, University of Iowa, Iowa City, Iowa, USA.

The purpose of this study was to characterize acute changes in inflammatory pathways in the mouse eye after blast-mediated traumatic brain injury (bTBI) and to determine whether modulation of these pathways could protect the structure and function of retinal ganglion cells (RGC). The bTBI was induced in C57BL/6J male mice by exposure to three 20 psi blast waves directed toward the head with the body shielded, with an inter-blast interval of one hour. Acute cytokine expression in retinal tissue was measured through reverse transcription-quantitative polymerase chain reaction (RT-qPCR) four hours post-blast. Increased retinal expression of ( and was observed in bTBI mice exposed to blast when compared with shams, which was associated with activation of microglia and macroglia reactivity, assessed via immunohistochemistry with ionized calcium binding adaptor molecule 1 and glial fibrillary acidic protein, respectively, one week post-blast. Blockade of the IL-1 pathway was accomplished using anakinra, an IL-1RI antagonist, administered intra-peritoneally for one week before injury and continuing for three weeks post-injury. Retinal function and RGC layer thickness were evaluated four weeks post-injury using pattern electroretinogram (PERG) and optical coherence tomography (OCT), respectively. After bTBI, anakinra treatment resulted in a preservation of RGC function and RGC structure when compared with saline treated bTBI mice. Optic nerve integrity analysis demonstrated a trend of decreased damage suggesting that IL-1 blockade also prevents axonal damage after blast. Blast exposure results in increased retinal inflammation including upregulation of pro-inflammatory cytokines and activation of resident microglia and macroglia. This may explain partially the RGC loss we observed in this model, as blockade of the acute inflammatory response after injury with the IL-1R1 antagonist anakinra resulted in preservation of RGC function and RGC layer thickness.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1089/neu.2019.6725DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7249480PMC
June 2020

Metabolite therapy guided by liquid biopsy proteomics delays retinal neurodegeneration.

EBioMedicine 2020 Feb 3;52:102636. Epub 2020 Feb 3.

Omics Laboratory, Department of Ophthalmology, Byers Eye Institute, Stanford University, Palo Alto, CA 94304, United States; Veterans Affairs Palo Alto Health Care System, Palo Alto, CA 94304, United States. Electronic address:

Background: Neurodegenerative diseases are incurable disorders caused by progressive neuronal cell death. Retinitis pigmentosa (RP) is a blinding neurodegenerative disease that results in photoreceptor death and progresses to the loss of the entire retinal network. We previously found that proteomic analysis of the adjacent vitreous served as way to indirectly biopsy the retina and identify changes in the retinal proteome.

Methods: We analyzed protein expression in liquid vitreous biopsies from autosomal recessive (ar)RP patients with PDE6A mutations and arRP mice with Pde6ɑ mutations. Proteomic analysis of retina and vitreous samples identified molecular pathways affected at the onset of photoreceptor death. Based on affected molecular pathways, arRP mice were treated with a ketogenic diet or metabolites involved in fatty-acid synthesis, oxidative phosphorylation, and the tricarboxylic acid (TCA) cycle.

Findings: Dietary supplementation of a single metabolite, ɑ-ketoglutarate, increased docosahexaeonic acid levels, provided neuroprotection, and enhanced visual function in arRP mice. A ketogenic diet delayed photoreceptor cell loss, while vitamin B supplementation had a limited effect. Finally, desorption electrospray ionization mass spectrometry imaging (DESI-MSI) on ɑ-ketoglutarate-treated mice revealed restoration of metabolites that correlated with our proteomic findings: uridine, dihydrouridine, and thymidine (pyrimidine and purine metabolism), glutamine and glutamate (glutamine/glutamate conversion), and succinic and aconitic acid (TCA cycle).

Interpretation: This study demonstrates that replenishing TCA cycle metabolites via oral supplementation prolongs retinal function and provides a neuroprotective effect on the photoreceptor cells and inner retinal network.

Funding: NIH grants [R01EY026682, R01EY024665, R01EY025225, R01EY024698, R21AG050437, P30EY026877, 5P30EY019007, R01EY018213, F30EYE027986, T32GM007337, 5P30CA013696], NSF grant CHE-1734082.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ebiom.2020.102636DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7005447PMC
February 2020

Fundoscopy-directed genetic testing to re-evaluate negative whole exome sequencing results.

Orphanet J Rare Dis 2020 01 30;15(1):32. Epub 2020 Jan 30.

Department of Ophthalmology, Columbia University, New York, NY, USA.

Background: Whole exome sequencing (WES) allows for an unbiased search of the genetic cause of a disease. Employing it as a first-tier genetic testing can be favored due to the associated lower incremental cost per diagnosis compared to when using it later in the diagnostic pathway. However, there are technical limitations of WES that can lead to inaccurate negative variant callings. Our study presents these limitations through a re-evaluation of negative WES results using subsequent tests primarily driven by fundoscopic findings. These tests included targeted gene testing, inherited retinal gene panels, whole genome sequencing (WGS), and array comparative genomic hybridization.

Results: Subsequent genetic testing guided by fundoscopy findings identified the following variant types causing retinitis pigmentosa that were not detected by WES: frameshift deletion and nonsense variants in the RPGR gene, 353-bp Alu repeat insertions in the MAK gene, and large exonic deletion variants in the EYS and PRPF31 genes. Deep intronic variants in the ABCA4 gene causing Stargardt disease and the GUCY2D gene causing Leber congenital amaurosis were also identified.

Conclusions: Negative WES analyses inconsistent with the phenotype should raise clinical suspicion. Subsequent genetic testing may detect genetic variants missed by WES and can make patients eligible for gene replacement therapy and upcoming clinical trials. When phenotypic findings support a genetic etiology, negative WES results should be followed by targeted gene sequencing, array based approach or whole genome sequencing.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s13023-020-1312-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6993391PMC
January 2020

Structural Insights into the Unique Activation Mechanisms of a Non-classical Calpain and Its Disease-Causing Variants.

Cell Rep 2020 01;30(3):881-892.e5

Omics Laboratory, Department of Ophthalmology, Byers Eye Institute, Stanford University, Palo Alto, CA 94304, USA; Veterans Affairs Palo Alto Health Care System, Palo Alto, CA 94304, USA. Electronic address:

Increased calpain activity is linked to neuroinflammation including a heritable retinal disease caused by hyper-activating mutations in the calcium-activated calpain-5 (CAPN5) protease. Although structures for classical calpains are known, the structure of CAPN5, a non-classical calpain, remains undetermined. Here we report the 2.8 Å crystal structure of the human CAPN5 protease core (CAPN5-PC). Compared to classical calpains, CAPN5-PC requires high calcium concentrations for maximal activity. Structure-based phylogenetic analysis and multiple sequence alignment reveal that CAPN5-PC contains three elongated flexible loops compared to its classical counterparts. The presence of a disease-causing mutation (c.799G>A, p.Gly267Ser) on the unique PC2L2 loop reveals a function in this region for regulating enzymatic activity. This mechanism could be transferred to distant calpains, using synthetic calpain hybrids, suggesting an evolutionary mechanism for fine-tuning calpain function by modifying flexible loops. Further, the open (inactive) conformation of CAPN5-PC provides structural insight into CAPN5-specific residues that can guide inhibitor design.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2019.12.077DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7001764PMC
January 2020

Novel mutations in the 3-box motif of the BACK domain of KLHL7 associated with nonsyndromic autosomal dominant retinitis pigmentosa.

Orphanet J Rare Dis 2019 12 19;14(1):295. Epub 2019 Dec 19.

Department of Ophthalmology, Columbia University Medical Center, New York, NY, USA.

Background: Mutations in the Kelch-like protein 7 (KLHL7) represent a recently described and, to date, poorly characterized etiology of inherited retinal dystrophy. Dominant mutations in KLHL7 are a cause of isolated, non-syndromic retinitis pigmentosa (RP). In contrast, recessive loss-of-function mutations are known to cause Crisponi or Bohring-Opitz like cold induced sweating syndrome-3 (BOS-3). In this study, the phenotype and progression of five unrelated patients with KLHL7 mediated autosomal dominant RP (adRP) are characterized. Clinical evaluation of these patients involved a complete ophthalmic exam, full-field electroretinography (ffERG), and imaging, including fundus photography, spectral domain optical coherence tomography (SD-OCT), short wavelength fundus autofluorescence (SW-AF), and near-infrared fundus autofluorescence (NIR-AF). Molecular diagnoses were performed using whole-exome sequencing or gene panel testing. Disease progression was monitored in three patients with available data for a mean follow up time of 4.5 ± 2.9 years. Protein modeling was performed for all variants found in this study in addition to those documented in the literature for recessive loss-of-function alleles causing Crisponi or Bohring-Opitz like cold-induced sweating syndrome.

Results: Genetic testing in three patients identified two novel variants within the 3-box motif of the BACK domain: c.472 T > C:p.(Cys158Arg) and c.433A > T:p.(Asn145Tyr). Clinical imaging demonstrated hyperautofluorescent ring formation on both SW-AF and NIR-AF in three patients, with diffuse peripheral and peripapillary atrophy seen in all but one case. SD-OCT demonstrated a phenotypic spectrum, from parafoveal atrophy of the outer retina with foveal sparing to widespread retinal thinning and loss of photoreceptors. Incidence of cystoid macular edema was high with four of five patients affected. Protein modeling of dominant alleles versus recessive loss-of-function alleles showed dominant alleles localized to the BTB and BACK domains while recessive alleles were found in the Kelch domain.

Conclusions: We report the phenotype in five patients with KLHL7 mediated adRP, two novel coding variants, and imaging biomarkers using SW-AF and NIR-AF. These findings may influence future gene-based therapies for adRP and pave the way for mechanistic studies that elucidate the pathogenesis of KLHL7-mediated RP.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s13023-019-1275-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6924004PMC
December 2019

Compound heterozygous novel frameshift variants in the gene result in Leber congenital amaurosis.

Cold Spring Harb Mol Case Stud 2019 12 13;5(6). Epub 2019 Dec 13.

Department of Ophthalmology, Columbia University, New York, New York 10019, USA.

The () gene encodes an 865-amino acid glycoprotein that is expressed in retinoblastoma cell lines and in the adult retina. The protein is localized to photoreceptor outer segment disc membranes, where it plays a structural role, and in the retinal pigment epithelium (RPE), where it acts as a cytosolic protein that mediates autophagy. Mutations in are typically associated with cone-rod dystrophy 12 (OMIM#3612657), autosomal dominant retinal macular dystrophy 2 (OMIM#608051), autosomal recessive retinitis pigmentosa 41 (OMIM#612095), and Stargardt disease 4 (OMIM#603786). Here we describe the first case of -associated Leber congenital amaurosis (LCA) in a 12-yr-old Asian male, caused by two not previously described deleterious frameshift variants in the compound heterozygous state. Clinical features include the presence of bull's eye maculopathy, pendular horizontal nystagmus, and photodysphoria consistent with the clinical diagnosis of LCA. The patient was evaluated using ophthalmic imaging, electroretinography, and whole-exome sequencing. Electroretinography revealed extinguished retinal activity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/mcs.a004481DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6913139PMC
December 2019

A Reversible Silicon Oil-Induced Ocular Hypertension Model in Mice.

J Vis Exp 2019 11 15(153). Epub 2019 Nov 15.

Department of Ophthalmology, Stanford University School of Medicine;

Elevated intraocular pressure (IOP) is a well-documented risk factor for glaucoma. Here we describe a novel, effective method for consistently inducing stable IOP elevation in mice that mimics the post-operative complication of using silicone oil (SO) as a tamponade agent in human vitreoretinal surgery. In this protocol, SO is injected into the anterior chamber of the mouse eye to block the pupil and prevent inflow of aqueous humor. The posterior chamber accumulates aqueous humor and this in turn increases the IOP of the posterior segment. A single SO injection produces reliable, sufficient, and stable IOP elevation, which induces significant glaucomatous neurodegeneration. This model is a true replicate of secondary glaucoma in the eye clinic. To further mimic the clinical setting, SO can be removed from the anterior chamber to reopen the drainage pathway and allow inflow of aqueous humor, which is drained through the trabecular meshwork (TM) at the angle of the anterior chamber. Because IOP quickly returns to normal, the model can be used to test the effect of lowering IOP on glaucomatous retinal ganglion cells. This method is straightforward, does not require special equipment or repeat procedures, closely simulates clinical situations, and may be applicable to diverse animal species. However, minor modifications may be required.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3791/60409DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6938455PMC
November 2019

Novel REEP6 gene mutation associated with autosomal recessive retinitis pigmentosa.

Doc Ophthalmol 2020 02 19;140(1):67-75. Epub 2019 Sep 19.

Jonas Children's Vision Care, and Bernard & Shirlee Brown Glaucoma Laboratory, Columbia Stem Cell Initiative, Departments of Ophthalmology, Pathology and Cell Biology, Institute of Human Nutrition, Vagelos College of Physicians and Surgeons, Columbia University, New York, NY, USA.

Purpose: This study reports the ophthalmic and genetic findings of a Cameroonian patient with autosomal recessive retinitis pigmentosa (arRP) caused by a novel Receptor Expression Enhancing Protein 6 (REEP6) homozygous mutation.

Patient And Methods: A 33-year-old man underwent comprehensive ophthalmic examinations, including visual acuity measurements, dilated fundus imaging, electroretinography (ERG), and spectral-domain optical coherence tomography (SD-OCT). Short-wavelength fundus autofluorescence (SW-AF) and near-infrared fundus autofluorescence (NIR-AF) were also evaluated. Whole exome sequencing (WES) was used to identify potential pathogenic variants.

Results: Fundus examination revealed typical RP findings with additional temporal ten micron yellow dots. SD-OCT imaging revealed cystoid macular edema and perifoveal outer retinal atrophy with centrally preserved inner segment ellipsoid zone (EZ) bands. Hyperreflective spots were seen in the inner retinal layers. On SW-AF images, a hypoautofluorescent area in the perifoveal area was observed. NIR-AF imaging revealed an irregularly shaped hyperautofluorescent ring. His visual acuity was mildly affected. ERG showed undetectable rod responses and intact cone responses. Genetic testing via WES revealed a novel homozygous mutation (c.295G>A, p.Glu99Lys) in the gene encoding REEP6, which is predicted to alter the charge in the transmembrane helix.

Conclusions: This report is not only the first description of a Cameroonian patient with arRP associated with a REEP6 mutation, but also this particular genetic alteration. Substitution of p.Glu99Lys in REEP6 likely disrupts the interactions between REEP6 and the ER membrane. NIR-AF imaging may be particularly useful for assessing functional photoreceptor cells and show an "avocado" pattern of hyperautofluorescence in patients with the REEP6 mutation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s10633-019-09719-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7310602PMC
February 2020

Hypoxic drive caused type 3 neovascularization in a preclinical model of exudative age-related macular degeneration.

Hum Mol Genet 2019 10;28(20):3475-3485

Jonas Children's Vision Care and the Bernard & Shirlee Brown Glaucoma Laboratory, Herbert Irving Comprehensive Cancer Center, New York, NY 10032, USA.

Hypoxia associated with the high metabolic demand of rods has been implicated in the pathology of age-related macular degeneration (AMD), the most common cause of adult blindness in the developed world. The majority of AMD-associated severe vision loss cases are due to exudative AMD, characterized by neovascularization. To further investigate the causes and histopathology of exudative AMD, we conditionally induced hypoxia in a novel preclinical AMD model (Pde6gcreERT2/+;Vhl-/-) by targeting Vhl and used multimodal imaging and immunohistochemistry to track the development of hypoxia-induced neovascularization. In addition to developing a preclinical model that phenocopies exudative AMD, our studies revealed that the photoreceptor hypoxic response initiates and drives type 3 neovascularization, mainly in the outer retina. Activation of the VHL-HIF1a-VEGF-EPO pathway in the adult retina led to long-term neovascularization, retinal hemorrhages and compromised retinal layers. Our novel preclinical model would accelerate the testing of therapies that use metabolomic approaches to ameliorate AMD.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/hmg/ddz159DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7275777PMC
October 2019

CAPN5 genetic inactivation phenotype supports therapeutic inhibition trials.

Hum Mutat 2019 12 26;40(12):2377-2392. Epub 2019 Aug 26.

Omics Laboratory, Byers Eye Institute, Department of Ophthalmology, Stanford University, Palo Alto, California.

Small molecule pharmacological inhibition of dominant human genetic disease is a feasible treatment that does not rely on the development of individual, patient-specific gene therapy vectors. However, the consequences of protein inhibition as a clinical therapeutic are not well-studied. In advance of human therapeutic trials for CAPN5 vitreoretinopathy, genetic inactivation can be used to infer the effect of protein inhibition in vivo. We created a photoreceptor-specific knockout (KO) mouse for Capn5 and compared the retinal phenotype to both wild-type and an existing Capn5 KO mouse model. In humans, CAPN5 loss-of-function (LOF) gene variants were ascertained in large exome databases from 60,706 unrelated subjects without severe disease phenotypes. Ocular examination of the retina of Capn5 KO mice by histology and electroretinography showed no significant abnormalities. In humans, there were 22 LOF CAPN5 variants located throughout the gene and in all major protein domains. Structural modeling of coding variants showed these LOF variants were nearby known disease-causing variants within the proteolytic core and in regions of high homology between human CAPN5 and 150 homologs, yet the LOF of CAPN5 was tolerated as opposed to gain-of-function disease-causing variants. These results indicate that localized inhibition of CAPN5 is a viable strategy for hyperactivating disease alleles.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/humu.23894DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7493429PMC
December 2019

Opposing T cell responses in experimental autoimmune encephalomyelitis.

Nature 2019 08 7;572(7770):481-487. Epub 2019 Aug 7.

Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA, USA.

Experimental autoimmune encephalomyelitis is a model for multiple sclerosis. Here we show that induction generates successive waves of clonally expanded CD4, CD8 and γδ T cells in the blood and central nervous system, similar to gluten-challenge studies of patients with coeliac disease. We also find major expansions of CD8 T cells in patients with multiple sclerosis. In autoimmune encephalomyelitis, we find that most expanded CD4 T cells are specific for the inducing myelin peptide MOG. By contrast, surrogate peptides derived from a yeast peptide major histocompatibility complex library of some of the clonally expanded CD8 T cells inhibit disease by suppressing the proliferation of MOG-specific CD4 T cells. These results suggest that the induction of autoreactive CD4 T cells triggers an opposing mobilization of regulatory CD8 T cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41586-019-1467-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7145319PMC
August 2019

Comparison of structural progression between ciliopathy and non-ciliopathy associated with autosomal recessive retinitis pigmentosa.

Orphanet J Rare Dis 2019 08 1;14(1):187. Epub 2019 Aug 1.

Department of Ophthalmology, Columbia University, New York, NY, USA.

Background: To evaluate and compare the progression of ciliopathy and non-ciliopathy autosomal recessive Retinitis Pigmentosa patients (arRP) by measuring the constriction of hyperautofluorescent rings in fundus autofluorescence (FAF) images and the progressive shortening of the ellipsoid zone line width obtained by spectral-domain optical coherence tomography (SD-OCT).

Results: For the ciliopathy group, the estimated mean shortening of the ellipsoid zone line was 259 μm per year and the ring area decreased at a rate of 2.46 mm per year. For the non-ciliopathy group, the estimated mean shortening of the ellipsoid zone line was 84 μm per year and the ring area decreased at a rate of 0.7 mm per year.

Conclusions: Our study was able to quantify and compare the loss of EZ line width and short-wavelength autofluorescence (SW-AF) ring constriction progression over time for ciliopathy and non-ciliopathy arRP genes. These results may serve as a basis for modeling RP disease progression, and furthermore, they could potentially be used as endpoints in clinical trials seeking to promote cone and rod survival in RP patients.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s13023-019-1163-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6676605PMC
August 2019

CRISPR Base Editing in Induced Pluripotent Stem Cells.

Methods Mol Biol 2019 ;2045:337-346

Department of Ophthalmology, Columbia University, New York, NY, USA.

Induced pluripotent stem cells (iPSCs) have demonstrated tremendous potential in numerous disease modeling and regenerative medicine-based therapies. The development of innovative gene transduction and editing technologies has further augmented the potential of iPSCs. Cas9-cytidine deaminases, for example, have developed as an alternative strategy to integrate single-base mutations (C → T or G → A transitions) at specific genomic loci. In this chapter, we specifically describe CRISPR (clustered regularly interspaced short palindromic repeats) base editing in iPSCs for editing precise locations in the genome. This state-of-the-art approach enables highly efficient and accurate modifications in genes. Thus, this technique not only has the potential to have biotechnology and therapeutic applications but also the ability to reveal underlying mechanisms regarding pathologies caused by specific mutations.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/7651_2019_243DOI Listing
June 2020