Publications by authors named "Venkata R M Chavali"

22 Publications

  • Page 1 of 1

The Role of Genetic Ancestry as a Risk Factor for Primary Open-angle Glaucoma in African Americans.

Invest Ophthalmol Vis Sci 2021 Feb;62(2):28

Scheie Eye Institute, University of Pennsylvania, Philadelphia, Pennsylvania, United States.

Purpose: POAG is the leading cause of irreversible blindness in African Americans. In this study, we quantitatively assess the association of autosomal ancestry with POAG risk in a large cohort of self-identified African Americans.

Methods: Subjects recruited to the Primary Open-Angle African American Glaucoma Genetics (POAAGG) study were classified as glaucoma cases or controls by fellowship-trained glaucoma specialists. POAAGG subjects were genotyped using the MEGA Ex array (discovery cohort, n = 3830; replication cohort, n = 2135). Population structure was interrogated using principal component analysis in the context of the 1000 Genomes Project superpopulations.

Results: The majority of POAAGG samples lie on an axis between African and European superpopulations, with great variation in admixture. Cases had a significantly lower mean value of the ancestral component q0 than controls for both cohorts (P = 6.14-4; P = 3-6), consistent with higher degree of African ancestry. Among POAG cases, higher African ancestry was also associated with thinner central corneal thickness (P = 2-4). Admixture mapping showed that local genetic ancestry was not a significant risk factor for POAG. A polygenic risk score, comprised of 23 glaucoma-associated single nucleotide polymorphisms from the NHGRI-EBI genome-wide association study catalog, was significant in both cohorts (P < 0.001), suggesting that both known POAG single nucleotide polymorphisms and an omnigenic ancestry effect influence POAG risk.

Conclusions: In sum, the POAAGG study population is very admixed, with a higher degree of African ancestry associated with an increased POAG risk. Further analyses should consider social and environmental factors as possible confounding factors for disease predisposition.
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http://dx.doi.org/10.1167/iovs.62.2.28DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7900887PMC
February 2021

Exome-wide evaluation of rare coding variants using electronic health records identifies new gene-phenotype associations.

Nat Med 2021 01 11;27(1):66-72. Epub 2021 Jan 11.

Department of Genetics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.

The clinical impact of rare loss-of-function variants has yet to be determined for most genes. Integration of DNA sequencing data with electronic health records (EHRs) could enhance our understanding of the contribution of rare genetic variation to human disease. By leveraging 10,900 whole-exome sequences linked to EHR data in the Penn Medicine Biobank, we addressed the association of the cumulative effects of rare predicted loss-of-function variants for each individual gene on human disease on an exome-wide scale, as assessed using a set of diverse EHR phenotypes. After discovering 97 genes with exome-by-phenome-wide significant phenotype associations (P < 10), we replicated 26 of these in the Penn Medicine Biobank, as well as in three other medical biobanks and the population-based UK Biobank. Of these 26 genes, five had associations that have been previously reported and represented positive controls, whereas 21 had phenotype associations not previously reported, among which were genes implicated in glaucoma, aortic ectasia, diabetes mellitus, muscular dystrophy and hearing loss. These findings show the value of aggregating rare predicted loss-of-function variants into 'gene burdens' for identifying new gene-disease associations using EHR phenotypes in a medical biobank. We suggest that application of this approach to even larger numbers of individuals will provide the statistical power required to uncover unexplored relationships between rare genetic variation and disease phenotypes.
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http://dx.doi.org/10.1038/s41591-020-1133-8DOI Listing
January 2021

Short prolactin isoforms are expressed in photoreceptors of canine retinas undergoing retinal degeneration.

Sci Rep 2021 Jan 11;11(1):460. Epub 2021 Jan 11.

Division of Experimental Retinal Therapies, Section of Ophthalmology, Department of Clinical Sciences and Advanced Medicine, School of Veterinary Medicine, University of Pennsylvania, 3900 Delancey Street, Philadelphia, PA, 19104, USA.

Prolactin (PRL) hormone functions as a pleiotropic cytokine with a protective role in the retina. We recently identified by transcriptome profiling that PRL is one of the most highly upregulated mRNAs in the retinas of mutant rcd1 (PDE6B) and xlpra2 (RPGR) dogs at advanced stages of photoreceptor disease. In the present study, we have identified the expression of a short PRL isoform that lacks exon 1 in canine retinas and analyzed the time-course of expression and localization of this isoform in the retinas of these two models. Using laser capture microdissection to isolate RNA from each of the retinal cellular layers, we found by qPCR that this short PRL isoform is expressed in photoreceptors of degenerating retinas. We confirmed by in situ hybridization that its expression is localized to the outer nuclear layer and begins shortly after the onset of disease at the time of peak photoreceptor cell death in both models. PRL protein was also detected only in mutant dog retinas. Our results call for further investigations into the role of this novel PRL isoform in retinal degeneration.
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http://dx.doi.org/10.1038/s41598-020-80691-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7801730PMC
January 2021

Molecular Genetics and Functional Analysis Implicate Involvement in POAG Pathogenesis.

Cells 2020 08 21;9(9). Epub 2020 Aug 21.

Scheie Eye Institute, Department of Ophthalmology, Philadelphia, PA 19104, USA.

The genes in the 9p21 locus ( & ) are widely associated with Primary open-angle glaucoma (POAG). However, the functional importance of this locus in POAG pathogenesis is still unexplored. This study investigated the role of axis in POAG. We observed significant association of SNP rs4977756 with POAG and its endophenotypic traits (vertical cup-disc ratio ( = 0.033) and central corneal thickness ( 0.008)) by screening African American POAG cases ( = 1567) and controls ( = 1600). A luciferase reporter assay in Human embryonic kidney 293T (HEK293T) cells revealed that the region surrounding rs4977756 likely serves as a transcriptional repressor. siRNA-mediated knockdown of in HEK293T cells and trabecular meshwork (TM) cells resulted in significantly increased expression of , which was also observed in human POAG ocular tissues. Pathway focused qRT-PCR gene expression analysis showed increased cellular senescence, TGFβ signaling and ECM deposition in TM cells after suppression. In conclusion, we report that may act as a regulator, and it could function by modulating the expression of . In addition, increase in CDKN2B levels due to suppression may result in the senescence of trabecular meshwork cells leading to POAG pathogenesis.
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http://dx.doi.org/10.3390/cells9091934DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7564117PMC
August 2020

Dual SMAD inhibition and Wnt inhibition enable efficient and reproducible differentiations of induced pluripotent stem cells into retinal ganglion cells.

Sci Rep 2020 07 16;10(1):11828. Epub 2020 Jul 16.

F.M. Kirby Center for Molecular Ophthalmology and Center for Advanced Retinal and Ocular Therapeutics (CAROT), 309B Stellar Chance Laboratories, Philadelphia, PA, 19104, USA.

Glaucoma is a group of progressive optic neuropathies that share common biological and clinical characteristics including irreversible changes to the optic nerve and visual field loss caused by the death of retinal ganglion cells (RGCs). The loss of RGCs manifests as characteristic cupping or optic nerve degeneration, resulting in visual field loss in patients with Glaucoma. Published studies on in vitro RGC differentiation from stem cells utilized classical RGC signaling pathways mimicking retinal development in vivo. Although many strategies allowed for the generation of RGCs, increased variability between experiments and lower yield hampered the cross comparison between individual lines and between experiments. To address this critical need, we developed a reproducible chemically defined in vitro methodology for generating retinal progenitor cell (RPC) populations from iPSCs, that are efficiently directed towards RGC lineage. Using this method, we reproducibly differentiated iPSCs into RGCs with greater than 80% purity, without any genetic modifications. We used small molecules and peptide modulators to inhibit BMP, TGF-β (SMAD), and canonical Wnt pathways that reduced variability between iPSC lines and yielded functional and mature iPSC-RGCs. Using CD90.2 antibody and Magnetic Activated Cell Sorter (MACS) technique, we successfully purified Thy-1 positive RGCs with nearly 95% purity.
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http://dx.doi.org/10.1038/s41598-020-68811-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7366935PMC
July 2020

Primary Open-Angle African American Glaucoma Genetics (POAAGG) Study: gender and risk of POAG in African Americans.

PLoS One 2019 1;14(8):e0218804. Epub 2019 Aug 1.

Scheie Eye Institute, University of Pennsylvania, Philadelphia, PA, United States of America.

The purpose of this study was to investigate the association between gender and primary open-angle glaucoma (POAG) among African Americans and to assess demographic, systemic, and behavioral factors that may contribute to differences between genders. The Primary Open-Angle African American Glaucoma Genetics (POAAGG) study had a case-control design and included African Americans 35 years and older, recruited from the greater Philadelphia, Pennsylvania. Diagnosis of POAG was based on evidence of both glaucomatous optic nerve damage and characteristic visual field loss. Demographic and behavioral information, history of systemic diseases and anthropometric measurements were obtained at study enrollment. Gender differences in risk of POAG were examined using multivariate logistic regression. A total of 2,290 POAG cases and 2,538 controls were included in the study. The percentage of men among cases was higher than among controls (38.6% vs 30.3%, P<0.001). The subjects' mean age at enrollment was significantly higher for cases compared to controls (70.2±11.3 vs. 61.6±11.8 years, P<0.003). Cases had lower rates of diabetes (40% vs. 46%, P<0.001), higher rates of systemic hypertension (80% vs. 72%, P<0.001), and lower body mass index (BMI) (29.7±6.7 vs. 31.9±7.4, P<0.001) than controls. In the final multivariable model, male gender was significantly associated with POAG risk (OR, 1.64; 95% CI, 1.44-1.87; P<0.001), after adjusting for age, systemic hypertension, diabetes, and BMI. Within the POAAGG study, men were at higher risk of having POAG than women. Pending genetic results from this study will be used to better understand the underlying genetic variations that may account for these differences.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0218804PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6675103PMC
March 2020

SNP located in an repeat downstream of rs4657473, is protective for POAG in African Americans.

Br J Ophthalmol 2019 10 12;103(10):1530-1536. Epub 2019 Mar 12.

Ophthalmology, Scheie Eye Institute, Philadelphia, Pennsylvania, USA.

Aims: To determine the association of single nucleotide polymorphisms (SNPs) downstream from the gene with primary open-angle glaucoma (POAG) in African Americans (AA).

Methods: AA subjects were recruited for the Primary Open-Angle African American Glaucoma Genetics (POAAGG) study from the Scheie Eye Institute and its satellite sites in Philadelphia. A region containing an repeat and seven SNPs, including rs4656461 near the gene, were PCR-Sanger sequenced from POAAGG cases (n=1537) and controls (n=1570). Association between POAG and SNPs near was investigated by logistic regression analysis. Phenotypic trait associations with these SNPs were assessed by analysis of variance. Electrophoretic mobility shift assay (EMSA) was performed to assess the affinity of human T-box 5 (TBX5) protein for a predicted binding motif in the region. Dual Luciferase assays were performed by transfecting recombinant plasmids containing the region surrounding the above SNPs in HEK293T and trabecular meshwork cells.

Results: The SNP rs4657473 (C>T) was associated with POAG; the TT genotype was protective (OR 0.20, 95% CI 0.09 to 0.42; p<0.001). No significant associations were found between the variants and phenotypic traits. EMSA confirmed the affinity of TBX5 for a predicted binding motif containing SNP rs4657475. Luciferase assays demonstrated a regulatory function for the genomic region around SNP rs4656561, located within repeat.

Conclusion: Our results demonstrate that a SNP downstream of TMCO1, rs4657473, is associated with POAG in an AA population. Our studies suggest a regulatory role for the previously POAG-associated locus near the gene that may affect gene expression.
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http://dx.doi.org/10.1136/bjophthalmol-2018-313086DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6817700PMC
October 2019

The association of mitochondrial DNA haplogroups with POAG in African Americans.

Exp Eye Res 2019 04 15;181:85-89. Epub 2019 Jan 15.

Scheie Eye Institute, University of Pennsylvania, Philadelphia, PA, USA. Electronic address:

Mitochondrial dysfunction has been implicated in the pathogenesis of primary open-angle glaucoma (POAG). However, the potential significance of mitochondrial DNA (mtDNA) haplogroups to POAG has not been evaluated in the overaffected African American population. To investigate the association of mtDNA haplogroups with POAG and its phenotypic characteristics, genotyping data from 4081 African American subjects (1919 cases and 2162 controls) was analyzed using 1293 positions on mtDNA. The overall frequency of mtDNA haplogroups in the Primary Open-Angle African American Glaucoma Genetics (POAAGG) study cohort was 37% L3, 29% L2, 21% L1, 4% L0, and 10% non-African haplogroups (non-L). When all haplogroups (L0, L1, L2, and non-L) were compared against theL3 reference group, after adjusting by age and principal component of ancestry, the non-L3 haplogroups showed higher risk of POAG (OR-1.19, p = 0.02), with a particularly strong association among males (OR = 1.41, p = 0.003). More specifically the non-L group was associated with higher POAG risk than the L3 haplogroup (OR = 1.77, p = 0.007, Bonferroni adjusted p = 0.027) and to the L3e (n = 256, OR = 1.92, p = 0.007, Bonferroni adjusted p = 0.029). No significant association was found when genders were analyzed together or in female only analysis. There were no significant differences in various POAG endophenotypes across mtDNA haplogroups. This study expands our knowledge of mitochondrial genetics and mtDNA haplogroup associations in African American POAG. Further work is needed to better understand the functional role of mtDNA polymorphisms and their interactions with nuclear genes that affect POAG.
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http://dx.doi.org/10.1016/j.exer.2019.01.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6443410PMC
April 2019

Whole-genome methylation profiling of the retinal pigment epithelium of individuals with age-related macular degeneration reveals differential methylation of the SKI, GTF2H4, and TNXB genes.

Clin Epigenetics 2019 01 14;11(1). Epub 2019 Jan 14.

Department of Eye and Vision Science, William Duncan Building, University of Liverpool, Liverpool, UK.

Background: Age-related macular degeneration (AMD) is a degenerative disorder of the central retina and the foremost cause of blindness. The retinal pigment epithelium (RPE) is a primary site of disease pathogenesis. The genetic basis of AMD is relatively well understood; however, this knowledge is yet to yield a treatment for the most prevalent non-neovascular disease forms. Therefore, tissue-specific epigenetic mechanisms of gene regulation are of considerable interest in AMD. We aimed to identify differentially methylated genes associated with AMD in the RPE and differentiate local DNA methylation aberrations from global DNA methylation changes, as local DNA methylation changes may be more amenable to therapeutic manipulation.

Methods: Epigenome-wide association study and targeted gene expression profiling were carried out in RPE cells from eyes of human donors. We performed genome-wide DNA methylation profiling (Illumina 450k BeadChip array) on RPE cells from 44 human donor eyes (25 AMD and 19 normal controls). We validated the findings using bisulfite pyrosequencing in 55 RPE samples (30 AMD and 25 normal controls) including technical (n = 38) and independent replicate samples (n = 17). Long interspersed nucleotide element 1 (LINE-1) analysis was then applied to assess global DNA methylation changes in the RPE. RT-qPCR on independent donor RPE samples was performed to assess gene expression changes.

Results: Genome-wide DNA methylation profiling identified differential methylation of multiple loci including the SKI proto-oncogene (SKI) (p = 1.18 × 10), general transcription factor IIH subunit H4 (GTF2H4) (p = 7.03 × 10), and Tenascin X (TNXB) (p = 6.30 × 10) genes in AMD. Bisulfite pyrosequencing validated the differentially methylated locus cg18934822 in SKI, and cg22508626 within GTF2H4, and excluded global DNA methylation changes in the RPE in AMD. We further demonstrated the differential expression of SKI, GTF2H4, and TNXB in the RPE of independent AMD donors.

Conclusions: We report the largest genome-wide methylation analysis of RPE in AMD along with associated gene expression changes to date, for the first-time reaching genome-wide significance, and identified novel targets for functional and future therapeutic intervention studies. The novel differentially methylated genes SKI and GTF2H4 have not been previously associated with AMD, and regulate disease pathways implicated in AMD, including TGF beta signaling (SKI) and transcription-dependent DNA repair mechanisms (GTF2H4).
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http://dx.doi.org/10.1186/s13148-019-0608-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6332695PMC
January 2019

The MT-CO1 V83I Polymorphism is a Risk Factor for Primary Open-Angle Glaucoma in African American Men.

Invest Ophthalmol Vis Sci 2018 04;59(5):1751-1759

Scheie Eye Institute, University of Pennsylvania, Philadelphia, Pennsylvania, United States.

Purpose: We investigate the function of the V83I polymorphism (m.6150G>A, rs879053914) in the mitochondrial cytochrome c oxidase subunit 1 (MT-CO1) gene and its role in African American (AA) primary open-angle glaucoma (POAG).

Methods: This study used Sanger sequencing (1339 cases, 850 controls), phenotypic characterization of Primary Open-Angle African American Glaucoma Genetics study (POAAGG) cases, a masked chart review of CO1 missense cases (V83I plus M117T, n = 29) versus wild type cases (n = 29), a yeast 2-hybrid (Y2H) cDNA library screen, and quantification of protein-protein interactions by Y2H and ELISA.

Results: The association of V83I with POAG in AA was highly significant for men (odds ratio [OR] 6.5; 95% confidence interval [CI] 2.0-21.3, P = 0.0001), but not for women (OR 1.1; 95% CI, 0.62-2.00, P = 0.78). POAG cases having CO1 double missense mutation (V83I + M117T, L1c2 haplogroup) had a higher cup-to-disc ratio (0.77 vs. 0.71, P = 0.04) and significantly worse visual function (average pattern standard deviation, 6.5 vs. 4.3, P = 0.009; average mean deviation -10.4 vs. -4.5, P = 0.006) when compared to matched wild type cases (L1b haplogroup). Interaction of the V83I region of CO1 with amyloid beta peptide (Aβ) was confirmed by ELISA assay, and this interaction was abrogated by V83I. A Y2H screen of an adult human brain cDNA library with the V83 region of CO1 as bait retrieved the UBQLN1 gene.

Conclusions: The V83I polymorphism was associated strongly with POAG in AA men and disrupts Aβ-binding to CO1. This region also interacts with a neuroprotective protein, UBQLN1.
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http://dx.doi.org/10.1167/iovs.17-23277DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5886029PMC
April 2018

Characterizing the "POAGome": A bioinformatics-driven approach to primary open-angle glaucoma.

Prog Retin Eye Res 2017 05 20;58:89-114. Epub 2017 Feb 20.

Scheie Eye Institute, University of Pennsylvania, Philadelphia, PA, 19104, USA.

Primary open-angle glaucoma (POAG) is a genetically, physiologically, and phenotypically complex neurodegenerative disorder. This study addressed the expanding collection of genes associated with POAG, referred to as the "POAGome." We used bioinformatics tools to perform an extensive, systematic literature search and compiled 542 genes with confirmed associations with POAG and its related phenotypes (normal tension glaucoma, ocular hypertension, juvenile open-angle glaucoma, and primary congenital glaucoma). The genes were classified according to their associated ocular tissues and phenotypes, and functional annotation and pathway analyses were subsequently performed. Our study reveals that no single molecular pathway can encompass the pathophysiology of POAG. The analyses suggested that inflammation and senescence may play pivotal roles in both the development and perpetuation of the retinal ganglion cell degeneration seen in POAG. The TGF-β signaling pathway was repeatedly implicated in our analyses, suggesting that it may be an important contributor to the manifestation of POAG in the anterior and posterior segments of the globe. We propose a molecular model of POAG revolving around TGF-β signaling, which incorporates the roles of inflammation and senescence in this disease. Finally, we highlight emerging molecular therapies that show promise for treating POAG.
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http://dx.doi.org/10.1016/j.preteyeres.2017.02.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5464971PMC
May 2017

Presence of rd8 mutation does not alter the ocular phenotype of late-onset retinal degeneration mouse model.

Mol Vis 2015 13;21:273-84. Epub 2015 Mar 13.

Shiley Eye Institute, University of California San Diego, La Jolla, CA.

Purpose: A spontaneous frameshift mutation, c.3481delC, in the Crb1 gene is the underlying cause of dysplasia and retinal degeneration in rd8 mice. The rd8 mutation is found in C57BL/6N but not in C57BL/6J mouse sub-strains. The development of ocular pathology in single knockout Ccl2-/-, Cx3cr1-/- and in double knockout Ccl2-/-, Cx3cr1-/- mice raised on a C57BL/6 background has been reported to depend on the presence of a rd8 mutation. In this study, we investigated the influence of the rd8 mutation on the retinal pathology that we previously described in the late-onset retinal degeneration (L-ORD) mouse model with a heterozygous S163R mutation in the C1q-tumor necrosis factor-related protein-5Ctrp5+/- gene that was generated on a C57BL/6J background.

Methods: Mouse lines carrying the Ctrp5 S163R and rd8 mutations (Ctrp5+/-;rd8/rd8), corresponding controls without the rd8 mutation (Ctrp5+/-;wt/wt), and wild-type mice with and without the rd8 mutation (Wtrd8/rd8 and Wtwt/wt, respectively) were generated by systematic breeding of mice in our L-ORD mouse colony. Genotyping the mice for the rd8 (del C at nt3481 in Crb1) and Ctrp5 S163R mutations was performed with allelic PCR or sequencing. Retinal morphology was studied with fundus imaging, histology, light microscopy, electron microscopy, and immunohistochemistry.

Results: Genotype analysis of the mice in L-ORD mouse colony detected the rd8 mutation in the homozygous and heterozygous state. Fundus imaging of wild-type mice without the rd8 mutation (Wtwt/wt) revealed no autofluorescence (AF) spots up to 6-8 months and few AF spots at 21 months. However, the accumulation of AF lesions accelerated with age in the Ctrp5+/- mice that lack the rd8 mutation (Ctrp5+/-;wt/wt). The number of AF lesions was significantly increased (p<0.001), and they were small and uniformly distributed throughout the retina in the 21-month-old Ctrp5+/-;wt/wt mice when compared to the age-matched controls. Wild-type and Ctrp5+/- mice with the rd8 mutation (Wtrd8/rd8 and Ctrp5+/-;rd8/rd8, respectively) revealed an integrated retinal architecture with well-defined outer segments/inner segments (OS/IS), outer nuclear layer (ONL), outer plexiform layer (OPL), and inner nuclear layer (INL). The presence of pseudorosette structures reported in the rd8 mice between the ONL and the INL in the ventral quadrant of the retina was not observed in all genotypes studied. Further, the external limiting membrane was continuous in the Ctrp5+/-;rd8/rd8 and Wtrd8/rd8 mice. Evaluation of the retinal phenotype revealed that the Ctrp5+/-;wt/wt mice developed characteristic L-ORD pathology including age-dependent accumulation of AF spots, development of sub-retinal, sub-RPE, and basal laminar deposits, and Bruch's membrane abnormalities at older age, while these changes were not observed in the age-matched littermate WTwt/wt mice.

Conclusions: The Wtrd8/rd8 and Ctrp5+/-;rd8/rd8 mice raised on C57BL/6J did not develop early onset retinal changes that are characteristic of the rd8 phenotype, supporting the hypothesis that manifestation of rd8-associated pathology depends on the genetic background. The retinal pathology observed in mice with the Ctrp5+/-;wt/wt genotype is consistent with the L-ORD phenotype observed in patients and with the phenotype we described previously. The lack of rd8-associated retinal pathology in the Ctrp5+/-;wt/wt mouse model raised on the C57BL/6J background and the development of the L-ORD phenotype in these mice in the presence and absence of the rd8 mutation suggests that the pathology observed in the Ctrp5+/-;wt/wt mice is primarily associated with the S163R mutation in the Ctrp5 gene.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4360165PMC
September 2015

Exome analysis identified a novel mutation in the RBP4 gene in a consanguineous pedigree with retinal dystrophy and developmental abnormalities.

PLoS One 2012 26;7(11):e50205. Epub 2012 Nov 26.

National Eye Institute, NIH, Bethesda, Maryland, United States of America.

Retinitis Pigmentosa (RP) is a common form of retinal degeneration characterized by photoreceptor degeneration and retinal pigment epithelium (RPE) atrophy causing loss of visual field and acuities. Exome sequencing identified a novel homozygous splice site variant (c.111+1G>A) in the gene encoding retinol binding protein 4 (RBP4). This change segregated with early onset, progressive, and severe autosomal recessive retinitis pigmentosa (arRP) in an eight member consanguineous pedigree of European ancestry. Additionally, one patient exhibited developmental abnormalities including patent ductus arteriosus and chorioretinal and iris colobomas. The second patient developed acne from young age and extending into the 5(th) decade. Both patients had undetectable levels of RBP4 in the serum suggesting that this mutation led to either mRNA or protein instability resulting in a null phenotype. In addition, the patients exhibited severe vitamin A deficiency, and diminished serum retinol levels. Circulating transthyretin levels were normal. This study identifies the RBP4 splice site change as the cause of RP in this pedigree. The presence of developmental abnormalities and severe acne in patients with retinal degeneration may indicate the involvement of genes that regulate vitamin A absorption, transport and metabolism.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0050205PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3506607PMC
May 2013

Cloning, characterization, and expression analysis of the pig (Sus scrofa) C1q tumor necrosis factor-related protein-5 gene.

Mol Vis 2012 17;18:92-102. Epub 2012 Jan 17.

Department of Animal Science, North Carolina State University, Raleigh, NC, USA.

Purpose: Autosomal dominant early-onset long anterior zonules (LAZs) and late-onset retinal degeneration (L-ORD) in humans are associated with the S163R mutation of the complement 1q-tumor necrosis factor related protein-5 (CTRP5) gene. For using the pig as an L-ORD model for the study of pathology, we cloned, characterized, and studied the expression profile of pig CTRP5 (pCTRP5).

Methods: The pCTRP5 was cloned and sequenced from porcine genomic DNA. Bioinformatic analysis was done to evaluate the functional domains present in the pCTRP5 using PROSITE tools. The V5 epitope-tagged constructs of pCTRP5 and the mammalian promoters, elongation factor 1-α (EF) promoter and 579 bp of the putative promoter located upstream to pCTRP5 DNA, were used for in vitro expression analysis. The pCTRP5 expression, protein size, and cellular localization were studied in transiently transfected Cos-7 or ARPE-19 cells by western blot analysis using anti-CTRP5 and anti-V5 epitope antibodies. Expression of pCTRP5 in the pig eye tissues was analyzed by western blot analysis, real-time PCR, and immunohistochemistry.

Results: As predicted, pCTRP5 showed a 92% DNA homology and 98% amino acid homology with human CTRP5 (hCTRP5). Bioinformatic analysis revealed the presence of an alternate in-frame translational start site upstream to the presumed initiator codon. The presence of a putative promoter region upstream to the pCTRP5 was identified. The putative pCTRP5 promoter was found to be functional by western blot analysis. The size of the pCTRP5 protein (pCTRP5) was consistent with its predicted molecular weight, indicating that the potential alternative start site was not used. Western blot and RT-PCR analyses showed that pCTRP5 was predominantly expressed in RPE, a pattern of expression consistent with that found in mouse and human eyes.

Conclusions: The sequence and genomic organization of pCTRP5 was found to be similar to the human homolog. The DNA and protein sequence of pCTRP5 are highly homologous to hCTRP5, indicating that they are highly conserved. A putative promoter region (579 bp) present upstream to pCTRP5 was found to be functional and was able to drive the expression of the pCTRP5 gene cloned downstream. The tissue distribution in the eye and the expression profile of pCTRP5 in transiently transfected cells is consistent with hCTRP5 expression. Immunohistochemistry analysis of the pig retinal sections revealed localization of pCTRP5 to the apical and basolateral regions on the RPE and in the ciliary body. The potential in-frame alternate start site was found to be nonfunctional by western blot analysis of transiently transfected cells. Similarities between human and pig CTRP5 and the presence of an area centralis region in the pig similar to the human macula, together with its large eyeball size, makes the domestic pig a good model for the study of LAZs and L-ORD.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3265177PMC
September 2012

GNAT1 associated with autosomal recessive congenital stationary night blindness.

Invest Ophthalmol Vis Sci 2012 Mar 13;53(3):1353-61. Epub 2012 Mar 13.

National Centre of Excellence in Molecular Biology, University of the Punjab, Lahore, Pakistan.

Purpose: Congenital stationary night blindness is a nonprogressive retinal disorder manifesting as impaired night vision and is generally associated with other ocular symptoms, such as nystagmus, myopia, and strabismus. This study was conducted to further investigate the genetic basis of CSNB in a consanguineous Pakistani family.

Methods: A consanguineous family with multiple individuals manifesting cardinal symptoms of congenital stationary night blindness was ascertained. All family members underwent detailed ophthalmic examination, including fundus photographic examination and electroretinography. Blood samples were collected and genomic DNA was extracted. Exclusion and genome-wide linkage analyses were completed and two-point LOD scores were calculated. Bidirectional sequencing of GNAT1 was completed, and quantitative expression of Gnat1 transcript levels were investigated in ocular tissues at different postnatal intervals.

Results: The results of ophthalmic examinations were suggestive of early-onset stationary night blindness with no extraocular anomalies. The genome-wide scan localized the critical interval to chromosome 3, region p22.1-p14.3, with maximum two-point LOD scores of 3.09 at θ = 0, flanked by markers D3S3522 and D3S1289. Subsequently, a missense mutation in GNAT1, p.D129G, was identified, which segregated within the family, consistent with an autosomal recessive mode of inheritance, and was not present in 192 ethnically matched control chromosomes. Expression analysis suggested that Gnat1 is expressed at approximately postnatal day (P)7 and is predominantly expressed in the retina.

Conclusions: These data suggest that a homozygous missense mutation in GNAT1 is associated with autosomal recessive stationary night blindness.
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http://dx.doi.org/10.1167/iovs.11-8026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3339909PMC
March 2012

Rescue of photoreceptor degeneration by curcumin in transgenic rats with P23H rhodopsin mutation.

PLoS One 2011 29;6(6):e21193. Epub 2011 Jun 29.

Jacobs Retina Center, Department of Ophthalmology, University of California San Diego, La Jolla, California, United States of America.

The P23H mutation in the rhodopsin gene causes rhodopsin misfolding, altered trafficking and formation of insoluble aggregates leading to photoreceptor degeneration and autosomal dominant retinitis pigmentosa (RP). There are no effective therapies to treat this condition. Compounds that enhance dissociation of protein aggregates may be of value in developing new treatments for such diseases. Anti-protein aggregating activity of curcumin has been reported earlier. In this study we present that treatment of COS-7 cells expressing mutant rhodopsin with curcumin results in dissociation of mutant protein aggregates and decreases endoplasmic reticulum stress. Furthermore we demonstrate that administration of curcumin to P23H-rhodopsin transgenic rats improves retinal morphology, physiology, gene expression and localization of rhodopsin. Our findings indicate that supplementation of curcumin improves retinal structure and function in P23H-rhodopsin transgenic rats. This data also suggest that curcumin may serve as a potential therapeutic agent in treating RP due to the P23H rhodopsin mutation and perhaps other degenerative diseases caused by protein trafficking defects.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0021193PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3126808PMC
December 2011

A CTRP5 gene S163R mutation knock-in mouse model for late-onset retinal degeneration.

Hum Mol Genet 2011 May 24;20(10):2000-14. Epub 2011 Feb 24.

Ophthalmology, University of California San Diego, La Jolla, CA 92037, USA.

Late-onset retinal macular degeneration (L-ORD) is an autosomal dominant inherited disorder caused by a single missense mutation (S163R) in the CTRP5/C1QTNF5 protein. Early phenotypic features of L-ORD include: dark adaptation abnormalities, nyctalopia, and drusen deposits in the peripheral macular region. Apart from posterior segment abnormalities, these patients also develop abnormally long anterior lens zonules. In the sixth decade of life the rod and cone function declines, accompanied by electroretinogram (ERG) abnormalities. Some patients also develop choroidal neovascularization and glaucoma. In order to understand the disease pathology and mechanisms involved in retinal dystrophy, we generated a knock-in (Ctrp5(+/-)) mouse model carrying the disease-associated mutation in the mouse Ctrp5/C1QTNF5 gene. These mice develop slower rod-b wave recovery consistent with early dark adaptation abnormalities, accumulation of hyperautofluorescence spots, retinal pigment epithelium abnormalities, drusen, Bruch's membrane abnormalities, loss of photoreceptors, and retinal vascular leakage. The Ctrp5(+/-) mice, which have most of the pathological features of age-related macular degeneration, are unique and may serve as a valuable model both to understand the molecular pathology of late-onset retinal degeneration and to evaluate therapies.
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http://dx.doi.org/10.1093/hmg/ddr080DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3080610PMC
May 2011

A mutation in SLC24A1 implicated in autosomal-recessive congenital stationary night blindness.

Am J Hum Genet 2010 Oct 16;87(4):523-31. Epub 2010 Sep 16.

National Centre of Excellence in Molecular Biology, University of the Punjab, Lahore 53700, Pakistan.

Congenital stationary night blindness (CSNB) is a nonprogressive retinal disorder that can be associated with impaired night vision. The last decade has witnessed huge progress in ophthalmic genetics, including the identification of three genes implicated in the pathogenicity of autosomal-recessive CSNB. However, not all patients studied could be associated with mutations in these genes and thus other genes certainly underlie this disorder. Here, we report a large multigeneration family with five affected individuals manifesting symptoms of night blindness. A genome-wide scan localized the disease interval to chromosome 15q, and recombination events in affected individuals refined the critical interval to a 10.41 cM (6.53 Mb) region that harbors SLC24A1, a member of the solute carrier protein superfamily. Sequencing of all the coding exons identified a 2 bp deletion in exon 2: c.1613_1614del, which is predicted to result in a frame shift that leads to premature termination of SLC24A1 (p.F538CfsX23) and segregates with the disorder under an autosomal-recessive model. Expression analysis using mouse ocular tissues shows that Slc24a1 is expressed in the retina around postnatal day 7. In situ and immunohistological studies localized both SLC24A1 and Slc24a1 to the inner segment, outer and inner nuclear layers, and ganglion cells of the retina, respectively. Our data expand the genetic basis of CSNB and highlight the indispensible function of SLC24A1 in retinal function and/or maintenance in humans.
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http://dx.doi.org/10.1016/j.ajhg.2010.08.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2948789PMC
October 2010

A mutation in ZNF513, a putative regulator of photoreceptor development, causes autosomal-recessive retinitis pigmentosa.

Am J Hum Genet 2010 Sep;87(3):400-9

Ophthalmic Genetics and Visual Function Branch, National Eye Institute, National Institutes of Health, Bethesda, MD 20892, USA.

Retinitis pigmentosa (RP) is a phenotypically and genetically heterogeneous group of inherited retinal degenerations characterized clinically by night blindness, progressive constriction of the visual fields, and loss of vision, and pathologically by progressive loss of rod and then cone photoreceptors. Autosomal-recessive RP (arRP) in a consanguineous Pakistani family previously linked to chromosome 2p22.3-p24.1 is shown to result from a homozygous missense mutation (c.1015T>C [p.C339R]) in ZNF513, encoding a presumptive transcription factor. znf513 is expressed in the retina, especially in the outer nuclear layer, inner nuclear layer, and photoreceptors. Knockdown of znf513 in zebrafish reduces eye size, retinal thickness, and expression of rod and cone opsins and causes specific loss of photoreceptors. These effects are rescued by coinjection with wild-type (WT) but not p.C339R-znf513 mRNA. Both normal and p.C339R mutant ZNF513 proteins expressed in COS-7 cells localize to the nucleus. ChIP analysis shows that only the wild-type but not the mutant ZNF513 binds to the Pax6, Sp4, Arr3, Irbp, and photoreceptor opsin promoters. These results suggest that the ZNF513 p.C339R mutation is responsible for RP in this family and that ZNF513 plays a key role in the regulation of photoreceptor-specific genes in retinal development and photoreceptor maintenance.
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http://dx.doi.org/10.1016/j.ajhg.2010.08.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2933346PMC
September 2010

Identification of a promoter for the human C1Q-tumor necrosis factor-related protein-5 gene associated with late-onset retinal degeneration.

Invest Ophthalmol Vis Sci 2010 Nov 16;51(11):5499-507. Epub 2010 Jun 16.

Department of Ophthalmology, University of California San Diego, La Jolla, California, USA.

Purpose: The Complement-1q tumor necrosis factor-related protein 5 (C1QTNF5/CTRP5) gene is located in the 3' untranslated region of the Membrane Frizzled Related Protein (MFRP) gene, and these two genes are reported to be dicistronic. The authors examined the 5' upstream sequence of CTRP5 for the presence of a promoter regulating the expression of this gene.

Methods: The sequence upstream of the translational start site of human CTRP5 (hCTRP5) was analyzed by Promoter Inspector software. A series of plasmids containing segments of hCTRP5 putative promoter sequence (-29 bp to -3.6 kb) upstream of the luciferase gene were generated. Cells were transiently transfected with these plasmids, and luciferase activity was measured. 5' RACE analysis was performed to determine the functional transcription start site. V5 tagged-pig CTRP5 (pCTRP5) gene, cloned downstream of the hCTRP5 putative promoter, was expressed in a human retinal cell line (ARPE-19) and a Chinese hamster ovary cell line (CHO-K1) to study the functionality of the putative promoter.

Results: Bioinformatic analysis identified a putative promoter region between nt -1322 and +1 sequence of hCTRP5. 5' RACE analysis revealed the presence of the transcriptional start site (TSS) at 62 bp upstream of the start codon in the CTRP5. The 1.3-kb sequence of the hCTRP5 predicted promoter produced higher levels of luciferase activity, indicating the strength of the cloned CTRP5 promoter. The promoter sequence between nt -1322 bp to -29 bp upstream of the first ATG of CTRP5 was found to be essential for this promoter activity. The predicted hCTRP5 promoter was found to control the expression of V5-tagged pCTRP5 and nuclear GFP, indicating that the promoter was functional.

Conclusions: This study revealed the presence of a functional promoter for the CTRP5 gene located 5' of its start site. Understanding the regulation of CTRP5 gene transcription may provide insights into the possible role of CTRP5 in the retina and the pathology underlying late-onset retinal degeneration caused by mutations in this gene. In addition, these studies will determine whether CTRP5 and MFRP are functionally dicistronic.
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http://dx.doi.org/10.1167/iovs.10-5543DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3061496PMC
November 2010

Age-related retinal degeneration (arrd2) in a novel mouse model due to a nonsense mutation in the Mdm1 gene.

Hum Mol Genet 2008 Dec 18;17(24):3929-41. Epub 2008 Sep 18.

The Jackson Laboratory, Bar Harbor, ME 04609, USA.

We observed that a naturally occurring mouse strain developed age-related retinal degeneration (arrd2). These mice had normal fundi, electroretinograms (ERGs) and retinal histology at 6 months of age; vessel attenuation, RPE atrophy and pigmentary abnormalities at 14 months, which progressed to complete loss of photoreceptors and extinguished ERG by 22 months. Genetic analysis revealed that the retinal degeneration in arrd2 segregates in an autosomal recessive manner and the disease gene localizes to mouse chromosome 10. A positional candidate cloning approach detected a nonsense mutation in the mouse double minute-1 gene (Mdm1), which results in the truncation of the putative protein from 718 amino acids to 398. We have identified a novel transcript of the Mdm1 gene, which is the predominant transcript in the retina. The Mdm1 transcript is localized to the nuclear layers of neural retina. Expression of Mdm1 in the retina increases steadily from post-natal day 30 to 1 year, and a high level of Mdm1 are subsequently maintained. The Mdm1 transcript was found to be significantly depleted in the retina of arrd2 mice and the transcript was observed to degrade by nonsense-mediated decay. These results indicate that the depletion of the Mdm1 transcript may underlie the mechanism leading to late-onset progressive retinal degeneration in arrd2 mice. Analysis of a cohort of patients with age-related macular degeneration (AMD) wherein the susceptibility locus maps to chromosome 12q, a region bearing the human ortholog to MDM1, did not reveal association between human MDM1 and AMD.
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http://dx.doi.org/10.1093/hmg/ddn295DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2638579PMC
December 2008

Genome segment 6 of Antheraea mylitta cypovirus encodes a structural protein with ATPase activity.

Virology 2008 Jul 16;377(1):7-18. Epub 2008 May 16.

Department of Biotechnology, Indian Institute of Technology, Kharagpur, Kharagpur 721302, West Bengal, India.

The genome segment 6 (S6) of the 11 double stranded RNA genomes from Antheraea mylitta cypovirus was converted into cDNA, cloned and sequenced. S6 consisted of 1944 nucleotides with an ORF of 607 amino acids and could encode a protein of 68 kDa, termed P68. Motif scan and molecular docking analysis of P68 showed the presence of two cystathionine beta synthase (CBS) domains and ATP binding sites. The ORF of AmCPV S6 was expressed in E. coli as His-tag fusion protein and polyclonal antibody was raised. Immunoblot analysis of virus infected gut cells and purified polyhedra using raised anti-p68 polyclonal antibody showed that S6 encodes a viral structural protein. Fluorescence and ATPase assay of soluble P68 produced in Sf-9 cells via baculovirus expression system showed its ability to bind and cleave ATP. These results suggest that P68 may bind viral RNA through CBS domains and help in replication and transcription through ATP binding and hydrolysis.
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http://dx.doi.org/10.1016/j.virol.2008.03.038DOI Listing
July 2008