Academic Appointments

Administrative Appointments

  • Regular Member, NIH Biology of the Visual System Study Section (2017 - 2021)
  • Board of Scientific Counselors, National Eye Institute-NIH (2008 - 2013)

Boards, Advisory Committees, Professional Organizations

  • Scientific Advisory Board, Foundation Fighting Blindness (2009 - Present)

Professional Education

  • BS, University of Wisconsin-Madison, Biochemistry (1981)
  • PhD, Stanford University, Biochemistry (1988)
  • MD, Stanford University, Medicine (1989)

Current Research and Scholarly Interests

Work in the Vollrath laboratory is focused on understanding processes in the eye that are relevant to human health and disease. The eye is an organ particularly amenable to genetic analysis because its accessibility facilitates detection and characterization of a variety of disease states, yet such diseases rarely impair life span or fertility. We frequently start with genes known to be important in the eye because of their association with ocular disease phenotypes, and then investigate molecular mechanisms by uncovering pathways and processes relevant to normal eye function and pathogenesis.

RPE mitochondrial dysfunction is thought to play a causative role in retinal degenerative diseases such as mitochondrial retinopathy and age-related macular degeneration. As a test of this hypothesis, we generated mice with an RPE-selective postnatal loss of mitochondrial oxidative phosphorylation (OXPHOS). OXPHOS-deficient RPE cells are surprisingly long-lived, but lose critical epithelial characteristics through cellular dedifferentiation and, later, an epithelial to mesenchymal-like transition. OXPHOS-deficient RPE cells initiate a stress response that includes dependence upon the HGF/c-Met pathway, upregulation of aerobic glycolysis, activation of the mTOR signaling pathway, and cellular hypertrophy. Activation of mTOR and subsequent dedifferentiation can also be triggered by acute chemical oxidative damage to the RPE in vivo. For both chronic metabolic and acute oxidative RPE stress, the consequences for adjacent photoreceptors are profoundly negative, resulting in a gradual or rapid (respectively) retinal degeneration. Strikingly, treatment of animals with the mTOR inhibitor, rapamycin, blunts RPE dedifferentiation and hypertrophy and preserves photoreceptor numbers and function for both stressors. We would like to understand the mechanism of mTOR-mediated RPE dedifferentiation and determine whether this new in vivo RPE stress response is activated in human retinal disease.

Phagocytosis is an example of a basic process that we study. Every morning in mammalian eyes, the distal portion of the light sensing outer segments of photoreceptors are phagocytized by adjacent cells of the retinal pigment epithelium (RPE). Phagocytosis is balanced by new synthesis at the proximal end of the outer segment. Together, these two processes lead to constant turnover of outer segments and serve to repair light- and oxygen-induced damage. The daily ‘big breakfast’ of outer segment material, summed over the life of an animal, distinguishes the post-mitotic RPE cell as the most phagocytic cell in the body. Photoreceptor degeneration in mutant rats and mice with defective RPE phagocytosis demonstrates that this process is essential for the normal functioning of the mammalian retina. By genetic analysis of these mutant rodents, we identified the receptor tyrosine kinase, MERTK, as a critical part of the phagocytic mechanism. We also identified mutations in the human MERTK gene in individuals with a retinal degenerative disease known as retinitis pigmentosa. We have elaborated our understanding of the mechanism of phagocytosis by demonstrating that MERTK acts locally, at the site of phagocytosis, to promote ingestion of bound outer segment tips. MERTK does so by triggering a striking redistribution of myosin II from the cell periphery to sites of ingestion. We are continuing to investigate the mechanism of RPE phagocytosis with an emphasis on identifying new protein components and understanding its circadian regulation.

2023-24 Courses

Graduate and Fellowship Programs

All Publications

  • Alternative oxidase blunts pseudohypoxia and photoreceptor degeneration due to RPE mitochondrial dysfunction. Proceedings of the National Academy of Sciences of the United States of America Chen, M., Wang, Y., Dalal, R., Du, J., Vollrath, D. 2024; 121 (25): e2402384121


    Loss of mitochondrial electron transport complex (ETC) function in the retinal pigment epithelium (RPE) in vivo results in RPE dedifferentiation and progressive photoreceptor degeneration, and has been implicated in the pathogenesis of age-related macular degeneration. Xenogenic expression of alternative oxidases in mammalian cells and tissues mitigates phenotypes arising from some mitochondrial electron transport defects, but can exacerbate others. We expressed an alternative oxidase from Ciona intestinalis (AOX) in ETC-deficient murine RPE in vivo to assess the retinal consequences of stimulating coenzyme Q oxidation and respiration without ATP generation. RPE-restricted expression of AOX in this context is surprisingly beneficial. This focused intervention mitigates RPE mTORC1 activation, dedifferentiation, hypertrophy, stress marker expression, pseudohypoxia, and aerobic glycolysis. These RPE cell autonomous changes are accompanied by increased glucose delivery to photoreceptors with attendant improvements in photoreceptor structure and function. RPE-restricted AOX expression normalizes accumulated levels of succinate and 2-hydroxyglutarate in ETC-deficient RPE, and counteracts deficiencies in numerous neural retinal metabolites. These features can be attributed to the activation of mitochondrial inner membrane flavoproteins such as succinate dehydrogenase and proline dehydrogenase, and alleviation of inhibition of 2-oxyglutarate-dependent dioxygenases such as prolyl hydroxylases and epigenetic modifiers. Our work underscores the importance to outer retinal health of coenzyme Q oxidation in the RPE and identifies a metabolic network critical for photoreceptor survival in the context of RPE mitochondrial dysfunction.

    View details for DOI 10.1073/pnas.2402384121

    View details for PubMedID 38865272

  • In the Eyes of the Beholder-New Mertk Knockout Mouse and Re-Evaluation of Phagocytosis versus Anti-Inflammatory Functions of MERTK. International journal of molecular sciences Ghosh, S., Finnemann, S. C., Vollrath, D., Rothlin, C. V. 2024; 25 (10)


    Greg Lemke's laboratory was one of the pioneers of research into the TAM family of receptor tyrosine kinases (RTKs). Not only was Tyro3 cloned in his laboratory, but his group also extensively studied mice knocked out for individual or various combinations of the TAM RTKs Tyro3, Axl, and Mertk. Here we primarily focus on one of the paralogs-MERTK. We provide a historical perspective on rodent models of loss of Mertk function and their association with retinal degeneration and blindness. We describe later studies employing mouse genetics and the generation of newer knockout models that point out incongruencies with the inference that loss of MERTK-dependent phagocytosis is sufficient for severe, early-onset photoreceptor degeneration in mice. This discussion is meant to raise awareness with regards to the limitations of the original Mertk knockout mouse model generated using 129 derived embryonic stem cells and carrying 129 derived alleles and the role of these alleles in modifying Mertk knockout phenotypes or even displaying Mertk-independent phenotypes. We also suggest molecular approaches that can further Greg Lemke's scintillating legacy of dissecting the molecular functions of MERTK-a protein that has been described to function in phagocytosis as well as in the negative regulation of inflammation.

    View details for DOI 10.3390/ijms25105299

    View details for PubMedID 38791338

  • Therapeutic blood-brain barrier modulation and stroke treatment by a bioengineered FZD4-selective WNT surrogate in mice. Nature communications Ding, J., Lee, S., Vlahos, L., Yuki, K., Rada, C. C., van Unen, V., Vuppalapaty, M., Chen, H., Sura, A., McCormick, A. K., Tomaske, M., Alwahabi, S., Nguyen, H., Nowatzke, W., Kim, L., Kelly, L., Vollrath, D., Califano, A., Yeh, W., Li, Y., Kuo, C. J. 2023; 14 (1): 2947


    Derangements of the blood-brain barrier (BBB) or blood-retinal barrier (BRB) occur in disorders ranging from stroke, cancer, diabetic retinopathy, and Alzheimer's disease. The Norrin/FZD4/TSPAN12 pathway activates WNT/beta-catenin signaling, which is essential for BBB and BRB function. However, systemic pharmacologic FZD4 stimulation is hindered by obligate palmitoylation and insolubility of native WNTs and suboptimal properties of the FZD4-selective ligand Norrin. Here, we develop L6-F4-2, a non-lipidated, FZD4-specific surrogate which significantly improves subpicomolar affinity versus native Norrin. In Norrin knockout (NdpKO) mice, L6-F4-2 not only potently reverses neonatal retinal angiogenesis deficits, but also restores BRB and BBB function. In adult C57Bl/6J mice, post-stroke systemic delivery of L6-F4-2 strongly reduces BBB permeability, infarction, and edema, while improving neurologic score and capillary pericyte coverage. Our findings reveal systemic efficacy of a bioengineered FZD4-selective WNT surrogate during ischemic BBB dysfunction, with potential applicability to adult CNS disorders characterized by an aberrant blood-brain barrier.

    View details for DOI 10.1038/s41467-023-37689-1

    View details for PubMedID 37268690

  • Cilia-associated wound repair mediated by IFT88 in retinal pigment epithelium. Scientific reports Ning, K., Bhuckory, M. B., Lo, C. H., Sendayen, B. E., Kowal, T. J., Chen, M., Bansal, R., Chang, K. C., Vollrath, D., Berbari, N. F., Mahajan, V. B., Hu, Y., Sun, Y. 2023; 13 (1): 8205


    Primary cilia are conserved organelles that integrate extracellular cues into intracellular signals and are critical for diverse processes, including cellular development and repair responses. Deficits in ciliary function cause multisystemic human diseases known as ciliopathies. In the eye, atrophy of the retinal pigment epithelium (RPE) is a common feature of many ciliopathies. However, the roles of RPE cilia in vivo remain poorly understood. In this study, we first found that mouse RPE cells only transiently form primary cilia. We then examined the RPE in the mouse model of Bardet-Biedl Syndrome 4 (BBS4), a ciliopathy associated with retinal degeneration in humans, and found that ciliation in BBS4 mutant RPE cells is disrupted early during development. Next, using a laser-induced injury model in vivo, we found that primary cilia in RPE reassemble in response to laser injury during RPE wound healing and then rapidly disassemble after the repair is completed. Finally, we demonstrated that RPE-specific depletion of primary cilia in a conditional mouse model of cilia loss promoted wound healing and enhanced cell proliferation. In summary, our data suggest that RPE cilia contribute to both retinal development and repair and provide insights into potential therapeutic targets for more common RPE degenerative diseases.

    View details for DOI 10.1038/s41598-023-35099-3

    View details for PubMedID 37211572

    View details for PubMedCentralID 6513937

  • An efficient inducible RPE-Selective cre transgenic mouse line. Experimental eye research Chen, M., Kim, L., Lu, C. W., Zeng, H., Vollrath, D. 2020: 108370


    Cre-mediated modulation of gene function in the murine retinal pigment epithelium (RPE) has been widely used, but current postnatal RPE-selective Cre driver lines suffer from limited recombination efficiency and/or ectopic or mosaic expression. We sought to generate a transgenic mouse line with consistently efficient RPE-selective Cre activity that could be temporally regulated. We used phiC31 integrase to insert a DNA construct encoding a human BEST1 promoter fragment driving a Cre recombinase estrogen receptor fusion (BEST1-CreERT2) at the Rosa26 locus of C57BL/6J mice. Rosa26BEST1-CreERT2 mice were bred with a tdTomato reporter line and to mice with a Cre-conditional allele of Tfam. 4-hydroxytamoxifen or vehicle was delivered by four consecutive daily intraperitoneal injections. TdTomato was robustly expressed in the RPE of mice of both sexes for inductions beginning at P14 (males 90.7 ± 4.5%, females 84.7 ± 3.2%) and at 7 weeks (males 84.3 ± 7.0%, females 82 ± 3.6%). <0.6% of Muller glia also expressed tdTomato, but no tdTomato fluorescence was observed in other ocular cells or in multiple non-ocular tissues, with the exception of sparse foci in the testis. No evidence of retinal toxicity was observed in mice homozygous for the transgene induced beginning at P14 and assessed at 7-10 months. RPE-selective ablation of Tfam beginning at P14 led to reduced retinal thickness at 8 months of age and diminished retinal electrical responses at 12 months, as expected. These findings demonstrate that we have generated a mouse line with consistently efficient, tamoxifen-mediated postnatal induction of Cre recombination in the RPE and a small fraction of Muller glia. This line should be useful for temporally regulated modulation of gene function in the murine RPE.

    View details for DOI 10.1016/j.exer.2020.108370

    View details for PubMedID 33264655

  • AMP-independent activator of AMPK for treatment of mitochondrial disorders. PloS one Moore, T., Yanes, R. E., Calton, M. A., Vollrath, D., Enns, G. M., Cowan, T. M. 2020; 15 (10): e0240517


    Mitochondrial diseases are a clinically heterogenous group of disorders caused by respiratory chain dysfunction and associated with progressive, multi-systemic phenotype. There is no effective treatment or cure, and no FDA-approved drug for treating mitochondrial disease. To identify and characterize potential therapeutic compounds, we developed an in vitro screening assay and identified a group of direct AMP-activated protein kinase (AMPK) activators originally developed for the treatment of diabetes and metabolic syndrome. Unlike previously investigated AMPK agonists such as AICAR, these compounds allosterically activate AMPK in an AMP-independent manner, thereby increasing specificity and decreasing pleiotropic effects. The direct AMPK activator PT1 significantly improved mitochondrial function in assays of cellular respiration, energy status, and cellular redox. PT1 also protected against retinal degeneration in a mouse model of photoreceptor degeneration associated with mitochondrial dysfunction and oxidative stress, further supporting the therapeutic potential of AMP-independent AMPK agonists in the treatment of mitochondrial disease.

    View details for DOI 10.1371/journal.pone.0240517

    View details for PubMedID 33052980

  • Multi-trait genome-wide association study identifies new loci associated with optic disc parameters COMMUNICATIONS BIOLOGY Bonnemaijer, P. M., van Leeuwen, E. M., Iglesias, A. I., Gharahkhani, P., Vitart, V., Khawaja, A. P., Simcoe, M., Hoehn, R., Cree, A. J., Igo, R. P., Burdon, K. P., Craig, J. E., Hewitt, A. W., Jonas, J., Khor, C., Pasutto, F., Mackey, D. A., Mitchell, P., Mishra, A., Pang, C., Pasquale, L. R., Springelkamp, H., Thorleifsson, G., Thorsteinsdottir, U., Viswanathan, A. C., Wojciechowski, R., Wong, T., Young, T. L., Zeller, T., Atan, D., Aslam, T., Barman, S. A., Barrett, J. H., Bishop, P., Blows, P., Bunce, C., Carare, R. O., Chakravarthy, U., Chan, M., Chua, S. L., Crabb, D. P., Cumberland, P. M., Day, A., Desai, P., Dhillon, B., Dick, A. D., Egan, C., Ennis, S., Foster, P., Fruttiger, M., Gallacher, J. J., Garway, D. F., Gibson, J., Gore, D., Guggenheim, J. A., Hardcastle, A., Harding, S. P., Hogg, R. E., Keane, P. A., Khaw, P. T., Lascaratos, G., Macgillivray, T., Mackie, S., Martin, K., McGaughey, M., McGuinness, B., Mckay, G. J., McKibbin, M., Mitry, D., Moore, T., Morgan, J. E., Muthy, Z. A., O'Sullivan, E., Owen, C. G., Patel, P., Paterson, E., Peto, T., Petzold, A., Rahi, J. S., Rudnikca, A. R., Self, J., Sivaprasad, S., Steel, D., Stratton, I., Strouthidis, N., Sudlow, C., Thomas, D., Trucco, E., Tufail, A., Vernon, S. A., Viswanathan, A. C., Williams, C., Williams, K., Woodside, J. V., Yates, M. M., Yip, J., Zheng, Y., Allingham, R., Budenz, D., Bailey, J., Fingert, J., Gaasterland, D., Gaasterland, T., Haines, J. L., Hark, L., Hauser, M., Kang, J., Kraft, P., Lee, R., Lichter, P., Liu, Y., Moroi, S., Pasquale, L. R., Pericak, M., Realini, A., Rhee, D., Richards, J. R., Ritch, R., Scott, W. K., Singh, K., Sit, A., Vollrath, D., Weinreb, R., Wollstein, G., Wilmer, D., Gerhold-Ay, A., Nickels, S., Wilson, J. F., Hayward, C., Boutin, T. S., Polasek, O., Aung, T., Khor, C., Amin, N., Lotery, A. J., Wiggs, J. L., Cheng, C., Hysi, P. G., Hammond, C. J., Thiadens, A. J., MacGregor, S., Klaver, C. W., van Duijn, C. M., Int Glaucoma Genetics Consortium, Uk Biobank Eye Vision Consortium, NEIGHBORHOOD Consortium 2019; 2: 435


    A new avenue of mining published genome-wide association studies includes the joint analysis of related traits. The power of this approach depends on the genetic correlation of traits, which reflects the number of pleiotropic loci, i.e. genetic loci influencing multiple traits. Here, we applied new meta-analyses of optic nerve head (ONH) related traits implicated in primary open-angle glaucoma (POAG); intraocular pressure and central corneal thickness using Haplotype reference consortium imputations. We performed a multi-trait analysis of ONH parameters cup area, disc area and vertical cup-disc ratio. We uncover new variants; rs11158547 in PPP1R36-PLEKHG3 and rs1028727 near SERPINE3 at genome-wide significance that replicate in independent Asian cohorts imputed to 1000 Genomes. At this point, validation of these variants in POAG cohorts is hampered by the high degree of heterogeneity. Our results show that multi-trait analysis is a valid approach to identify novel pleiotropic variants for ONH.

    View details for DOI 10.1038/s42003-019-0634-9

    View details for Web of Science ID 000500297500001

    View details for PubMedID 31798171

    View details for PubMedCentralID PMC6881308

  • Depletion of Mitochondrial DNA in Differentiated Retinal Pigment Epithelial Cells. Scientific reports Hu, X., Calton, M. A., Tang, S., Vollrath, D. 2019; 9 (1): 15355


    We investigated the effects of treating differentiated retinal pigment epithelial (RPE) cells with didanosine (ddI), which is associated with retinopathy in individuals with HIV/AIDS. We hypothesized that such treatment would cause depletion of mitochondrial DNA and provide insight into the consequences of degradation of RPE mitochondrial function in aging and disease. Treatment of differentiated ARPE-19 or human primary RPE cells with 200M ddI for 6-24 days was not cytotoxic but caused up to 60% depletion of mitochondrial DNA, and a similar reduction in mitochondrial membrane potential and NDUFA9 protein abundance. Mitochondrial DNA-depleted RPE cells demonstrated enhanced aerobic glycolysis by extracellular flux analysis, increased AMP kinase activation, reduced mTOR activity, and increased resistance to cell death in response to treatment with the oxidant, sodium iodate. We conclude that ddI-mediated mitochondrial DNA depletion promotes a glycolytic shift in differentiated RPE cells and enhances resistance to oxidative damage. Our use of ddI treatment to induce progressive depletion of mitochondrial DNA in differentiated human RPE cells should be widely applicable for other studies aimed at understanding RPE mitochondrial dysfunction in aging and disease.

    View details for DOI 10.1038/s41598-019-51761-1

    View details for PubMedID 31653972

  • Association of a Primary Open-Angle Glaucoma Genetic Risk Score With Earlier Age at Diagnosis. JAMA ophthalmology Fan, B. J., Bailey, J. C., Igo, R. P., Kang, J. H., Boumenna, T., Brilliant, M. H., Budenz, D. L., Fingert, J. H., Gaasterland, T., Gaasterland, D., Hauser, M. A., Kraft, P., Lee, R. K., Lichter, P. R., Liu, Y., Moroi, S. E., Myers, J. S., Pericak-Vance, M. A., Realini, A., Rhee, D. J., Richards, J. E., Ritch, R., Schuman, J. S., Scott, W. K., Singh, K., Sit, A. J., Vollrath, D., Weinreb, R. N., Wollstein, G., Zack, D. J., Haines, J. L., Pasquale, L. R., Wiggs, J. L. 2019


    Importance: Genetic variants associated with primary open-angle glaucoma (POAG) are known to influence disease risk. However, the clinical effect of associated variants individually or in aggregate is not known. Genetic risk scores (GRS) examine the cumulative genetic load by combining individual genetic variants into a single measure, which is assumed to have a larger effect and increased power to detect relevant disease-related associations.Objective: To investigate if a GRS that comprised 12 POAG genetic risk variants is associated with age at disease diagnosis.Design, Setting, and Participants: A cross-sectional study included individuals with POAG and controls from the Glaucoma Genes and Environment (GLAUGEN) study and the National Eye Institute Glaucoma Human Genetics Collaboration (NEIGHBOR) study. A GRS was formulated using 12 variants known to be associated with POAG, and the alleles associated with increasing risk of POAG were aligned in the case-control sets. In case-only analyses, the association of the GRS with age at diagnosis was analyzed as an estimate of disease onset. Results from cohort-specific analyses were combined with meta-analysis. Data collection started in August 2012 for the NEIGHBOR cohort and in July 2008 for the GLAUGEN cohort and were analyzed starting in March 2018.Main Outcomes and Measures: Association of a 12 single-nucleotide polymorphism POAG GRS with age at diagnosis in individuals with POAG using linear regression.Results: The GLAUGEN study included 976 individuals with POAG and 1140 controls. The NEIGHBOR study included 2132 individuals with POAG and 2290 controls. For individuals with POAG, the mean (SD) age at diagnosis was 63.6 (9.8) years in the GLAUGEN cohort and 66.0 (13.7) years in the NEIGHBOR cohort. For controls, the mean (SD) age at enrollment was 65.5 (9.2) years in the GLAUGEN cohort and 68.9 (11.4) years in the NEIGHBOR cohort. All study participants were European white. The GRS was strongly associated with POAG risk in case-control analysis (odds ratio per 1-point increase in score=1.24; 95% CI, 1.21-1.27; P=3.4*10-66). In case-only analyses, each higher GRS unit was associated with a 0.36-year earlier age at diagnosis (beta=-0.36; 95% CI, -0.56 to -0.16; P=4.0*10-4). Individuals in the top 5% of the GRS had a mean (SD) age at diagnosis of 5.2 (12.8) years earlier than those in the bottom 5% GRS (61.4 [12.7] vs 66.6 [12.9] years; P=5.0*10-4).Conclusions and Relevance: A higher dose of POAG risk alleles was associated with an earlier age at glaucoma diagnosis. On average, individuals with POAG with the highest GRS had 5.2-year earlier age at diagnosis of disease. These results suggest that a GRS that comprised genetic variants associated with POAG could help identify patients with risk of earlier disease onset impacting screening and therapeutic strategies.

    View details for DOI 10.1001/jamaophthalmol.2019.3109

    View details for PubMedID 31436842

  • Highly Differentiated Human Fetal RPE Cultures Are Resistant to the Accumulation and Toxicity of Lipofuscin-Like Material. Investigative ophthalmology & visual science Zhang, Q., Presswalla, F., Calton, M., Charniga, C., Stern, J., Temple, S., Vollrath, D., Zacks, D. N., Ali, R. R., Thompson, D. A., Miller, J. M. 2019; 60 (10): 3468–79


    Purpose: The accumulation of undigestible autofluorescent material (UAM), termed lipofuscin in vivo, is a hallmark of aged RPE. Lipofuscin derives, in part, from the incomplete degradation of phagocytized photoreceptor outer segments (OS). Whether this accumulated waste is toxic is unclear. We therefore investigated the effects of UAM in highly differentiated human fetal RPE (hfRPE) cultures.Methods: Unmodified and photo-oxidized OS were fed daily to confluent cultures of ARPE-19 RPE or hfRPE. The emission spectrum, composition, and morphology of resulting UAM were measured and compared to in vivo lipofuscin. Effects of UAM on multiple RPE phenotypes were assessed.Results: Compared to ARPE-19, hfRPE were markedly less susceptible to UAM buildup. Accumulated UAM in hfRPE initially resembled the morphology of lipofuscin from AMD eyes, but compacted and shifted spectrum over time to resemble lipofuscin from healthy aged human RPE. UAM accumulation mildly reduced transepithelial electrical resistance, ketogenesis, certain RPE differentiation markers, and phagocytosis efficiency, while inducing senescence and rare, focal pockets of epithelial-mesenchymal transition. However, it had no effects on mitochondrial oxygen consumption rate, certain other RPE differentiation markers, secretion of drusen components or polarity markers, nor cell death.Conclusions: hfRPE demonstrates a remarkable resistance to UAM accumulation, suggesting mechanisms for efficient OS processing that may be lost in other RPE culture models. Furthermore, while UAM alters hfRPE phenotype, the effects are modest, consistent with conflicting reports in the literature on the toxicity of lipofuscin. Our results suggest that healthy RPE may adequately adapt to and tolerate lipofuscin accumulation.

    View details for DOI 10.1167/iovs.19-26690

    View details for PubMedID 31408109

  • Primary fetal RPE cultures resist accumulation and toxicity of lipofuscin-like material, and accumulated material can be further reduced by autophagy induction. Zhang, Q., Presswalla, F., McCusker, A., Charniga, C., Calton, M. A., Vollrath, D., Temple, S., Stern, J., Zacks, D. N., Thompson, D. A., Miller, J. ASSOC RESEARCH VISION OPHTHALMOLOGY INC. 2019
  • Genetic analyses of human fetal retinal pigment epithelium gene expression suggest ocular disease mechanisms. Communications biology Liu, B., Calton, M. A., Abell, N. S., Benchorin, G., Gloudemans, M. J., Chen, M., Hu, J., Li, X., Balliu, B., Bok, D., Montgomery, S. B., Vollrath, D. 2019; 2 (1): 186


    The retinal pigment epithelium (RPE) serves vital roles in ocular development and retinal homeostasis but has limited representation in large-scale functional genomics datasets. Understanding how common human genetic variants affect RPE gene expression could elucidate the sources of phenotypic variability in selected monogenic ocular diseases and pinpoint causal genes at genome-wide association study (GWAS) loci. We interrogated the genetics of gene expression of cultured human fetal RPE (fRPE) cells under two metabolic conditions and discovered hundreds of shared or condition-specific expression or splice quantitative trait loci (e/sQTLs). Co-localizations of fRPE e/sQTLs with age-related macular degeneration (AMD) and myopia GWAS data suggest new candidate genes, and mechanisms by which a common RDH5 allele contributes to both increased AMD risk and decreased myopia risk. Our study highlights the unique transcriptomic characteristics of fRPE and provides a resource to connect e/sQTLs in a critical ocular cell type to monogenic and complex eye disorders.

    View details for DOI 10.1038/s42003-019-0430-6

    View details for PubMedID 31925026

  • Myocilin Mutations in Patients With Normal-Tension Glaucoma JAMA OPHTHALMOLOGY Alward, W. M., van der Heide, C., Khanna, C. L., Roos, B. R., Sivaprasad, S., Kam, J., Ritch, R., Lotery, A., Igo, R. P., Bailey, J., Stone, E. M., Scheetz, T. E., Kwon, Y. H., Pasquale, L. R., Wiggs, J. L., Fingert, J. H., Cooke, J. N., Pasquale, L. R., Wiggs, J. L., Fingert, J. H., Allingham, R., Brilliant, M., Budenz, D., Bailey, J., Fingert, J., Gaasterland, D., Gaasterland, T., Haines, J. L., Hauser, M., Igo, R., Kang, J., Lee, R., Lichter, P., Liu, Y., Moroi, S., Pasquale, L. R., Pericak-Vance, M., Realini, A., Rhee, D., Richards, J. R., Ritch, R., Schuman, J., Scott, W. K., Singh, K., Sit, A., Vollrath, D., Wiggs, J. L., Weinreb, R. N., Wollstein, G., Zack, D., NEIGHBORHOOD Consortium 2019; 137 (5): 559–63


    Mutations in the myocilin (MYOC) gene are the most common molecularly defined cause of primary open-angle glaucoma that typically occurs in patients with high intraocular pressures (IOP). One MYOC mutation, p.Gln368Ter, has been associated with as many as 1.6% of primary open-angle glaucoma cases that had a mean maximum recorded IOP of 30 mm Hg. However, to our knowledge, the role of the p.Gln368Ter mutation in patients with normal-tension glaucoma (NTG) with an IOP of 21 mm Hg or lower has not been investigated.To evaluate the role of the p.Gln368Ter MYOC mutation in patients with NTG.In this case-control study of the prevalence of the p.Gln368Ter mutation in patients with NTG, cohort 1 was composed of 772 patients with NTG and 2152 controls from the United States (Iowa, Minnesota, and New York) and England and cohort 2 was composed of 561 patients with NTG and 2606 controls from the Massachusetts Eye and Ear Infirmary and the NEIGHBORHOOD consortium. Genotyping was conducted using real-time polymerase chain reaction that was confirmed with Sanger sequencing, the imputation of genome-wide association study data, or an analysis of whole-exome sequence data. Data analysis occurred between April 2007 and April 2018.Comparison of the frequency of the p.Gln368Ter MYOC mutation between NTG cases and controls with the Fisher exact test.Of 6091 total participants, 3346 (54.9%) were women and 5799 (95.2%) were white. We detected the p.Gln368Ter mutation in 7 of 772 patients with NTG (0.91%) and 7 of 2152 controls (0.33%) in cohort 1 (P = .03). In cohort 2, we detected the p.Gln368Ter mutation in 4 of 561 patients with NTG (0.71%) and 10 of 2606 controls (0.38%; P = .15). When the cohorts were analyzed as a group, the p.Gln368Ter mutation was associated with NTG (odds ratio, 2.3; 95% CI, 0.98-5.3; P = .04).In cohorts 1 and 2, the p.Gln368Ter mutation in MYOC was found in patients with IOPs that were 21 mm Hg or lower (NTG), although at a frequency that is lower than previously detected in patients with higher IOP. These data suggest that the p.Gln368Ter mutation may be associated with glaucoma in patients with normal IOPs as well as in patients with IOPs that are greater than 21 mm Hg.

    View details for DOI 10.1001/jamaophthalmol.2019.0005

    View details for Web of Science ID 000467503600021

    View details for PubMedID 30816940

  • Abnormal mTORC1 signaling leads to retinal pigment epithelium degeneration. Theranostics Huang, J., Gu, S., Chen, M., Zhang, S. J., Jiang, Z., Chen, X., Jiang, C., Liu, G., Radu, R. A., Sun, X., Vollrath, D., Du, J., Yan, B., Zhao, C. 2019; 9 (4): 1170-1180


    Retinal pigment epithelial (RPE) degeneration is potentially involved in the pathogenesis of several retinal degenerative diseases. mTORC1 signaling is shown as a crucial regulator of many biological processes and disease progression. In this study, we aimed at investigating the role of mTORC1 signaling in RPE degeneration. Methods: Western blots were conducted to detect mTORC1 expression pattern during RPE degeneration. Cre-loxP system was used to generate RPE-specific mTORC1 activation mice. Fundus, immunofluorescence staining, transmission electron microscopy, and targeted metabolomic analysis were conducted to determine the effects of mTORC1 activation on RPE degeneration in vivo. Electroretinography, spectral-domain optical coherence tomography, and histological experiments were conducted to determine the effects of mTORC1 activation on choroidal and retinal function in vivo. Results: RPE-specific activation of mTORC1 led to RPE degeneration as shown by the loss of RPE-specific marker, compromised cell junction integrity, and intracellular accumulation of lipid droplets. RPE degeneration further led to abnormal choroidal and retinal function. The inhibition of mTORC1 signaling with rapamycin could partially reverse RPE degeneration. Targeted metabolomics analysis further revealed that mTORC1 activation affected the metabolism of purine, carboxylic acid, and niacin in RPE. Conclusion: This study revealed that abnormal activation of mTORC1 signaling leads to RPE degeneration, which could provide a promising target for the treatment of RPE dysfunction-related diseases.

    View details for DOI 10.7150/thno.26281

    View details for PubMedID 30867823

    View details for PubMedCentralID PMC6401408

  • Cross-ancestry genome-wide association analysis of corneal thickness strengthens link between complex and Mendelian eye diseases (vol 9, 1864, 2018) NATURE COMMUNICATIONS Iglesias, A. I., Mishra, A., Vitart, V., Bykhovskaya, Y., Hoehn, R., Springelkamp, H., Cuellar-Partida, G., Gharahkhani, P., Bailey, J., Willoughby, C. E., Li, X., Yazar, S., Nag, A., Khawaja, A. P., Polasek, O., Siscovick, D., Mitchell, P., Tham, Y., Haines, J. L., Kearns, L. S., Hayward, C., Shi, Y., van Leeuwen, E. M., Taylor, K. D., Bonnemaijer, P., Rotter, J. I., Martin, N. G., Zeller, T., Mills, R. A., Souzeau, E., Staffieri, S. E., Jonas, J. B., Schmidtmann, I., Boutin, T., Kang, J. H., Lucas, S. M., Wong, T., Beutel, M. E., Wilson, J. F., Uitterlinden, A. G., Vithana, E. N., Foster, P. J., Hysi, P. G., Hewitt, A. W., Khor, C., Pasquale, L. R., Montgomery, G. W., Klaver, C. W., Aung, T., Pfeiffer, N., Mackey, D. A., Hammond, C. J., Cheng, C., Craig, J. E., Rabinowitz, Y. S., Wiggs, J. L., Burdon, K. P., van Duijn, C. M., MacGregor, S., Wang, J., Rochtchina, E., Attia, J., Scott, R., Holliday, E. G., Wong, T., Baird, P. N., Xie, J., Inouye, M., Viswanathan, A., Sim, X., Allingham, R., Brilliant, M. H., Budenz, D. L., Christen, W. G., Fingert, J., Friedman, D. S., Gaasterland, D., Gaasterland, T., Hauser, M. A., Kraft, P., Lee, R. K., Lichter, P. R., Liu, Y., Loomis, S. J., Moroi, S. E., Pericak-Vance, M. A., Realini, A., Richards, J. E., Schuman, J. S., Scott, W. K., Singh, K., Sit, A. J., Vollrath, D., Weinreb, R. N., Wollstein, G., Zack, D. J., Zhang, K., Donnelly, P., Barroso, I., Blackwell, J. M., Bramon, E., Brown, M. A., Casas, J. P., Corvin, A., Deloukas, P., Duncanson, A., Jankowski, J., Markus, H. S., Mathew, C. G., Palmer, C. A., Plomin, R., Rautanen, A., Sawcer, S. J., Trembath, R. C., Wood, N. W., Spencer, C. A., Band, G., Bellenguez, C., Freeman, C., Hellenthal, G., Giannoulatou, E., Pirinen, M., Pearson, R., Strange, A., Su, Z., Vukcevic, D., Langford, C., Hunt, S. E., Edkins, S., Gwilliam, R., Blackburn, H., Bumpstead, S. J., Dronov, S., Gillman, M., Gray, E., Hammond, N., Jayakumar, A., McCann, O. T., Liddle, J., Potter, S. C., Ravindrarajah, R., Ricketts, M., Waller, M., Weston, P., Widaa, S., Whittaker, P., Blue Mountains Eye Study-GWAS Grp, Neighborhood Consortium, Wellcome Trust Case Control 2019; 10: 155


    Emmanuelle Souzeau, who contributed to analysis of data, was inadvertently omitted from the author list in the originally published version of this Article. This has now been corrected in both the PDF and HTML versions of the Article.

    View details for DOI 10.1038/s41467-018-07819-1

    View details for Web of Science ID 000455103200001

    View details for PubMedID 30622277

    View details for PubMedCentralID PMC6325104

  • Abnormal mTORC1 signaling leads to retinal pigment epithelium degeneration THERANOSTICS Huang, J., Gu, S., Chen, M., Zhang, S., Jiang, Z., Chen, X., Jiang, C., Liu, G., Radu, R. A., Sun, X., Vollrath, D., Du, J., Yan, B., Zhao, C. 2019; 9 (4): 1170–80

    View details for DOI 10.7150/thno.26281

    View details for Web of Science ID 000457199500018

  • Method for measuring extracellular flux from intact polarized epithelial monolayers. Molecular vision Calton, M. A., Beaulieu, M. O., Benchorin, G., Vollrath, D. 2018; 24: 425-433


    The Seahorse XFp platform is widely used for metabolic assessment of cultured cells. Current methods require replating of cells into specialized plates. This is problematic for certain cell types, such as primary human fetal RPE (hfRPE) cells, which must be cultured for months to become properly differentiated. Our goal was to overcome this limitation by devising a method for assaying intact cell monolayers with the Seahorse XFp, without the need for replating.Primary hfRPE cells were differentiated by prolonged culture on filter inserts. Triangular sections of filters with differentiated cells attached were excised, transferred to XFp cell culture miniplate wells, immobilized at the bottoms, and subjected to mitochondrial stress tests. Replated cells were measured for comparison. Differentiated hfRPE cells were challenged or not with bovine photoreceptor outer segments (POS), and mitochondrial stress tests were performed 3.5 h later, after filter excision and transfer to assay plates.Differentiated hfRPE cells assayed following filter excision demonstrated increased maximal respiration, increased spare respiration capacity, and increased extracellular acidification rate (ECAR) relative to replated controls. hfRPE cells challenged with POS exhibited increased maximal respiration and spare capacity, with no apparent change in the ECAR, relative to untreated controls.We have developed a method to reproducibly assay intact, polarized monolayers of hfRPE cells with the Seahorse XFp platform and have shown that the method yields more robust metabolic measurements compared to standard methods and is suitable for assessing the consequences of prolonged perturbations of differentiated cells. We expect our approach to be useful for a variety of studies involving metabolic assessment of adherent cells cultured on filters.

    View details for PubMedID 30034209

    View details for PubMedCentralID PMC6031101

  • Testosterone Pathway Genetic Polymorphisms in Relation to Primary Open-Angle Glaucoma: analysis in Two Large Datasets INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE Bailey, J., Gharahkhani, P., Kang, J. H., Butkiewicz, M., Sullivan, D. A., Weinreb, R. N., Aschard, H., Allingham, R., Ashley-Koch, A., Lee, R. K., Moroi, S. E., Brilliant, M. H., Wollstein, G., Schuman, J. S., Fingert, J. H., Budenz, D. L., Realini, T., Gaasterland, T., Scott, W. K., Singh, K., Sit, A. J., Igo, R. P., Song, Y. E., Hark, L., Ritch, R., Rhee, D. J., Vollrath, D., Zack, D. J., Medeiros, F., Vajaranant, T. S., Chasman, D. I., Christen, W. G., Pericak-Vance, M. A., Liu, Y., Kraft, P., Richards, J. E., Rosner, B. A., Hauser, M. A., Craig, J. E., Burdon, K. P., Hewitt, A. W., Mackey, D. A., Haines, J. L., MacGregor, S., Wiggs, J. L., Pasquale, L. R., ANZRAG Consortium 2018; 59 (2): 629–36


    Sex hormones may be associated with primary open-angle glaucoma (POAG), although the mechanisms are unclear. We previously observed that gene variants involved with estrogen metabolism were collectively associated with POAG in women but not men; here we assessed gene variants related to testosterone metabolism collectively and POAG risk.We used two datasets: one from the United States (3853 cases and 33,480 controls) and another from Australia (1155 cases and 1992 controls). Both datasets contained densely called genotypes imputed to the 1000 Genomes reference panel. We used pathway- and gene-based approaches with Pathway Analysis by Randomization Incorporating Structure (PARIS) software to assess the overall association between a panel of single nucleotide polymorphisms (SNPs) in testosterone metabolism genes and POAG. In sex-stratified analyses, we evaluated POAG overall and POAG subtypes defined by maximum IOP (high-tension [HTG] or normal tension glaucoma [NTG]).In the US dataset, the SNP panel was not associated with POAG (permuted P = 0.77), although there was an association in the Australian sample (permuted P = 0.018). In both datasets, the SNP panel was associated with POAG in men (permuted P ≤ 0.033) and not women (permuted P ≥ 0.42), but in gene-based analyses, there was no consistency on the main genes responsible for these findings. In both datasets, the testosterone pathway association with HTG was significant (permuted P ≤ 0.011), but again, gene-based analyses showed no consistent driver gene associations.Collectively, testosterone metabolism pathway SNPs were consistently associated with the high-tension subtype of POAG in two datasets.

    View details for PubMedID 29392307

  • Genetic correlations between intraocular pressure, blood pressure and primary open-angle glaucoma: a multi-cohort analysis EUROPEAN JOURNAL OF HUMAN GENETICS Aschard, H., Kang, J. H., Iglesias, A. I., Hysi, P., Bailey, J., Khawaja, A. P., Allingham, R., Ashley-Koch, A., Lee, R. K., Moroi, S. E., Brilliant, M. H., Wollstein, G., Schuman, J. S., Fingert, J. H., Budenz, D. L., Realini, T., Gaasterland, T., Scott, W. K., Singh, K., Sit, A. J., Igo, R. P., Song, Y. E., Hark, L., Ritch, R., Rhee, D. J., Gulati, V., Haven, S., Vollrath, D., Zack, D. J., Medeiros, F., Weinreb, R. N., Cheng, C., Chasman, D. I., Christen, W. G., Pericak-Vance, M. A., Liu, Y., Kraft, P., Richards, J. E., Rosner, B. A., Hauser, M. A., Klaver, C. W., vanDuijn, C. M., Haines, J., Wiggs, J. L., Pasquale, L. R., Int Glaucoma Genetics Consortium 2017; 25 (11): 1261–67


    Primary open-angle glaucoma (POAG) is the most common chronic optic neuropathy worldwide. Epidemiological studies show a robust positive relation between intraocular pressure (IOP) and POAG and modest positive association between IOP and blood pressure (BP), while the relation between BP and POAG is controversial. The International Glaucoma Genetics Consortium (n=27 558), the International Consortium on Blood Pressure (n=69 395), and the National Eye Institute Glaucoma Human Genetics Collaboration Heritable Overall Operational Database (n=37 333), represent genome-wide data sets for IOP, BP traits and POAG, respectively. We formed genome-wide significant variant panels for IOP and diastolic BP and found a strong relation with POAG (odds ratio and 95% confidence interval: 1.18 (1.14-1.21), P=1.8 × 10-27) for the former trait but no association for the latter (P=0.93). Next, we used linkage disequilibrium (LD) score regression, to provide genome-wide estimates of correlation between traits without the need for additional phenotyping. We also compared our genome-wide estimate of heritability between IOP and BP to an estimate based solely on direct measures of these traits in the Erasmus Rucphen Family (ERF; n=2519) study using Sequential Oligogenic Linkage Analysis Routines (SOLAR). LD score regression revealed high genetic correlation between IOP and POAG (48.5%, P=2.1 × 10-5); however, genetic correlation between IOP and diastolic BP (P=0.86) and between diastolic BP and POAG (P=0.42) were negligible. Using SOLAR in the ERF study, we confirmed the minimal heritability between IOP and diastolic BP (P=0.63). Overall, IOP shares genetic basis with POAG, whereas BP has limited shared genetic correlation with IOP or POAG.

    View details for PubMedID 28853718

  • Assessment of Murine Retinal Function by Electroretinography BIO-PROTOCOL Benchorin, G., Calton, M. A., Beaulieu, M. O., Vollrath, D. 2017; 7 (7)


    The electroretinogram (ERG) is a sensitive and noninvasive method for testing retinal function. In this protocol, we describe a method for performing ERGs in mice. Contact lenses on the mouse cornea measure the electrical response to a light stimulus of photoreceptors and downstream retinal cells, and the collected data are analyzed to evaluate retinal function.

    View details for PubMedID 29177186

  • Age at natural menopause genetic risk score in relation to age at natural menopause and primary open-angle glaucoma in a US-based sample MENOPAUSE-THE JOURNAL OF THE NORTH AMERICAN MENOPAUSE SOCIETY Pasquale, L. R., Aschard, H., Kang, J. H., Bailey, J. N., Lindstrom, S., Chasman, D. I., Christen, W. G., Allingham, R. R., Ashley-Koch, A., Lee, R. K., Moroi, S. E., Brilliant, M. H., Wollstein, G., Schuman, J. S., Fingert, J., Budenz, D. L., Realini, T., Gaasterland, T., Gaasterland, D., Scott, W. K., Singh, K., Sit, A. J., Igo, R. P., Song, Y. E., Hark, L., Ritch, R., Rhee, D. J., Gulati, V., Havens, S., Vollrath, D., Zack, D. J., Medeiros, F., Weinreb, R. N., Pericak-Vance, M. A., Liu, Y., Kraft, P., Richards, J. E., Rosner, B. A., Hauser, M. A., Haines, J. L., Wiggs, J. L. 2017; 24 (2): 150-156


    Several attributes of female reproductive history, including age at natural menopause (ANM), have been related to primary open-angle glaucoma (POAG). We assembled 18 previously reported common genetic variants that predict ANM to determine their association with ANM or POAG.Using data from the Nurses' Health Study (7,143 women), we validated the ANM weighted genetic risk score in relation to self-reported ANM. Subsequently, to assess the relation with POAG, we used data from 2,160 female POAG cases and 29,110 controls in the National Eye Institute Glaucoma Human Genetics Collaboration Heritable Overall Operational Database (NEIGHBORHOOD), which consists of 8 datasets with imputed genotypes to 5.6+ million markers. Associations with POAG were assessed in each dataset, and site-specific results were meta-analyzed using the inverse weighted variance method.The genetic risk score was associated with self-reported ANM (P = 2.2 × 10) and predicted 4.8% of the variance in ANM. The ANM genetic risk score was not associated with POAG (Odds Ratio (OR) = 1.002; 95% Confidence Interval (CI): 0.998, 1.007; P = 0.28). No single genetic variant in the panel achieved nominal association with POAG (P ≥0.20). Compared to the middle 80 percent, there was also no association with the lowest 10 percentile or highest 90 percentile of genetic risk score with POAG (OR = 0.75; 95% CI: 0.47, 1.21; P = 0.23 and OR = 1.10; 95% CI: 0.72, 1.69; P = 0.65, respectively).A genetic risk score predicting 4.8% of ANM variation was not related to POAG; thus, genetic determinants of ANM are unlikely to explain the previously reported association between the two phenotypes.This is an open-access article distributed under the Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CCBY-NC-ND), where it is permissible to download and share thework provided it is properly cited. The work cannot be changed in any way or used commercially.

    View details for DOI 10.1097/GME.0000000000000741

    View details for Web of Science ID 000393758800006

  • Assessing the Association of Mitochondrial Genetic Variation With Primary Open-Angle Glaucoma Using Gene-Set Analyses. Investigative ophthalmology & visual science Khawaja, A. P., Cooke Bailey, J. N., Kang, J. H., Allingham, R. R., Hauser, M. A., Brilliant, M., Budenz, D. L., Christen, W. G., Fingert, J., Gaasterland, D., Gaasterland, T., Kraft, P., Lee, R. K., Lichter, P. R., Liu, Y., Medeiros, F., Moroi, S. E., Richards, J. E., Realini, T., Ritch, R., Schuman, J. S., Scott, W. K., Singh, K., Sit, A. J., Vollrath, D., Wollstein, G., Zack, D. J., Zhang, K., Pericak-Vance, M., Weinreb, R. N., Haines, J. L., Pasquale, L. R., Wiggs, J. L. 2016; 57 (11): 5046-5052


    Recent studies indicate that mitochondrial proteins may contribute to the pathogenesis of primary open-angle glaucoma (POAG). In this study, we examined the association between POAG and common variations in gene-encoding mitochondrial proteins.We examined genetic data from 3430 POAG cases and 3108 controls derived from the combination of the GLAUGEN and NEIGHBOR studies. We constructed biological-system coherent mitochondrial nuclear-encoded protein gene-sets by intersecting the MitoCarta database with the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. We examined the mitochondrial gene-sets for association with POAG and with normal-tension glaucoma (NTG) and high-tension glaucoma (HTG) subsets using Pathway Analysis by Randomization Incorporating Structure.We identified 22 KEGG pathways with significant mitochondrial protein-encoding gene enrichment, belonging to six general biological classes. Among the pathway classes, mitochondrial lipid metabolism was associated with POAG overall (P = 0.013) and with NTG (P = 0.0006), and mitochondrial carbohydrate metabolism was associated with NTG (P = 0.030). Examining the individual KEGG pathway mitochondrial gene-sets, fatty acid elongation and synthesis and degradation of ketone bodies, both lipid metabolism pathways, were significantly associated with POAG (P = 0.005 and P = 0.002, respectively) and NTG (P = 0.0004 and P < 0.0001, respectively). Butanoate metabolism, a carbohydrate metabolism pathway, was significantly associated with POAG (P = 0.004), NTG (P = 0.001), and HTG (P = 0.010).We present an effective approach for assessing the contributions of mitochondrial genetic variation to open-angle glaucoma. Our findings support a role for mitochondria in POAG pathogenesis and specifically point to lipid and carbohydrate metabolism pathways as being important.

    View details for DOI 10.1167/iovs.16-20017

    View details for PubMedID 27661856

  • A Common Variant in MIR182 Is Associated With Primary Open-Angle Glaucoma in the NEIGHBORHOOD Consortium INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE Liu, Y., Bailey, J. C., Helwa, I., Dismuke, W. M., Cai, J., Drewry, M., Brilliant, M. H., Budenz, D. L., Christen, W. G., Chasman, D. I., Fingert, J. H., Gaasterland, D., Gaasterland, T., Gordon, M. O., Igo, R. P., Kang, J. H., Kass, M. A., Kraft, P., Lee, R. K., Lichter, P., Moroi, S. E., Realini, A., Richards, J. E., Ritch, R., Schuman, J. S., Scott, W. K., Singh, K., Sit, A. J., Song, Y. E., Vollrath, D., Weinreb, R., Medeiros, F., Wollstein, G., Zack, D. J., Zhang, K., Pericak-Vance, M. A., Gonzalez, P., Stamer, W. D., Kuchtey, J., Kuchtey, R. W., Allingham, R. R., Hauser, M. A., Pasquale, L. R., Haines, J. L., Wiggs, J. L. 2016; 57 (10): 4528-4535
  • Treatment of retinitis pigmentosa due to MERTK mutations by ocular subretinal injection of adeno-associated virus gene vector: results of a phase I trial. Human genetics Ghazi, N. G., Abboud, E. B., Nowilaty, S. R., Alkuraya, H., Alhommadi, A., Cai, H., Hou, R., Deng, W., Boye, S. L., Almaghamsi, A., Al Saikhan, F., Al-Dhibi, H., Birch, D., Chung, C., Colak, D., LaVail, M. M., Vollrath, D., Erger, K., Wang, W., Conlon, T., Zhang, K., Hauswirth, W., Alkuraya, F. S. 2016; 135 (3): 327-343


    MERTK is an essential component of the signaling network that controls phagocytosis in retinal pigment epithelium (RPE), the loss of which results in photoreceptor degeneration. Previous proof-of-concept studies have demonstrated the efficacy of gene therapy using human MERTK (hMERTK) packaged into adeno-associated virus (AAV2) in treating RCS rats and mice with MERTK deficiency. The purpose of this study was to assess the safety of gene transfer via subretinal administration of rAAV2-VMD2-hMERTK in subjects with MERTK-associated retinitis pigmentosa (RP). After a preclinical phase confirming the safety of the study vector in monkeys, six patients (aged 14 to 54, mean 33.3 years) with MERTK-related RP and baseline visual acuity (VA) ranging from 20/50 to <20/6400 were entered in a phase I open-label, dose-escalation trial. One eye of each patient (the worse-seeing eye in five subjects) received a submacular injection of the viral vector, first at a dose of 150 µl (5.96 × 10(10)vg; 2 patients) and then 450 µl (17.88 × 10(10)vg; 4 patients). Patients were followed daily for 10 days at 30, 60, 90, 180, 270, 365, 540, and 730 days post-injection. Collected data included (1) full ophthalmologic examination including best-corrected VA, intraocular pressure, color fundus photographs, macular spectral domain optical coherence tomography and full-field stimulus threshold test (FST) in both the study and fellow eyes; (2) systemic safety data including CBC, liver and kidney function tests, coagulation profiles, urine analysis, AAV antibody titers, peripheral blood PCR and ASR measurement; and (3) listing of ophthalmological or systemic adverse effects. All patients completed the 2-year follow-up. Subretinal injection of rAAV2-VMD2-hMERTK was associated with acceptable ocular and systemic safety profiles based on 2-year follow-up. None of the patients developed complications that could be attributed to the gene vector with certainty. Postoperatively, one patient developed filamentary keratitis, and two patients developed progressive cataract. Of these two patients, one also developed transient subfoveal fluid after the injection as well as monocular oscillopsia. Two patients developed a rise in AAV antibodies, but neither patient was positive for rAAV vector genomes via PCR. Three patients also displayed measurable improved visual acuity in the treated eye following surgery, although the improvement was lost by 2 years in two of these patients. Gene therapy for MERTK-related RP using careful subretinal injection of rAAV2-VMD2-hMERTK is not associated with major side effects and may result in clinical improvement in a subset of patients.

    View details for DOI 10.1007/s00439-016-1637-y

    View details for PubMedID 26825853

  • Genome-wide association analysis identifies TXNRD2, ATXN2 and FOXC1 as susceptibility loci for primary open-angle glaucoma. Nature genetics Bailey, J. N., Loomis, S. J., Kang, J. H., Allingham, R. R., Gharahkhani, P., Khor, C. C., Burdon, K. P., Aschard, H., Chasman, D. I., Igo, R. P., Hysi, P. G., Glastonbury, C. A., Ashley-Koch, A., Brilliant, M., Brown, A. A., Budenz, D. L., Buil, A., Cheng, C., Choi, H., Christen, W. G., Curhan, G., De Vivo, I., Fingert, J. H., Foster, P. J., Fuchs, C., Gaasterland, D., Gaasterland, T., Hewitt, A. W., Hu, F., Hunter, D. J., Khawaja, A. P., Lee, R. K., Li, Z., Lichter, P. R., Mackey, D. A., McGuffin, P., Mitchell, P., Moroi, S. E., Perera, S. A., Pepper, K. W., Qi, Q., Realini, T., Richards, J. E., Ridker, P. M., Rimm, E., Ritch, R., Ritchie, M., Schuman, J. S., Scott, W. K., Singh, K., Sit, A. J., Song, Y. E., Tamimi, R. M., Topouzis, F., Viswanathan, A. C., Verma, S. S., Vollrath, D., Wang, J. J., Weisschuh, N., Wissinger, B., Wollstein, G., Wong, T. Y., Yaspan, B. L., Zack, D. J., Zhang, K., Study, E. E., Weinreb, R. N., Pericak-Vance, M. A., Small, K., Hammond, C. J., Aung, T., Liu, Y., Vithana, E. N., Macgregor, S., Craig, J. E., Kraft, P., Howell, G., Hauser, M. A., Pasquale, L. R., Haines, J. L., Wiggs, J. L. 2016; 48 (2): 189-194


    Primary open-angle glaucoma (POAG) is a leading cause of blindness worldwide. To identify new susceptibility loci, we performed meta-analysis on genome-wide association study (GWAS) results from eight independent studies from the United States (3,853 cases and 33,480 controls) and investigated the most significantly associated SNPs in two Australian studies (1,252 cases and 2,592 controls), three European studies (875 cases and 4,107 controls) and a Singaporean Chinese study (1,037 cases and 2,543 controls). A meta-analysis of the top SNPs identified three new associated loci: rs35934224[T] in TXNRD2 (odds ratio (OR) = 0.78, P = 4.05 × 10(-11)) encoding a mitochondrial protein required for redox homeostasis; rs7137828[T] in ATXN2 (OR = 1.17, P = 8.73 × 10(-10)); and rs2745572[A] upstream of FOXC1 (OR = 1.17, P = 1.76 × 10(-10)). Using RT-PCR and immunohistochemistry, we show TXNRD2 and ATXN2 expression in retinal ganglion cells and the optic nerve head. These results identify new pathways underlying POAG susceptibility and suggest new targets for preventative therapies.

    View details for DOI 10.1038/ng.3482

    View details for PubMedID 26752265

  • Gene Therapy for MERTK-Associated Retinal Degenerations. Advances in experimental medicine and biology LaVail, M. M., Yasumura, D., Matthes, M. T., Yang, H., Hauswirth, W. W., Deng, W., Vollrath, D. 2016; 854: 487-493


    MERTK-associated retinal degenerations are thought to have defects in phagocytosis of shed outer segment membranes by the retinal pigment epithelium (RPE), as do the rodent models of these diseases. We have subretinally injected an RPE-specific AAV2 vector, AAV2-VMD2-hMERTK, to determine whether this would provide long-term photoreceptor rescue in the RCS rat, which it did for up to 6.5 months, the longest time point examined. Moreover, we found phagosomes in the RPE in the rescued regions of RCS retinas soon after the onset of light. The same vector also had a major protective effect in Mertk-null mice, with a concomitant increase in ERG response amplitudes in the vector-injected eyes. These findings suggest that planned clinical trials with this vector will have a favorable outcome.

    View details for DOI 10.1007/978-3-319-17121-0_65

    View details for PubMedID 26427450

  • The mTOR Kinase Inhibitor INK128 Blunts Migration of Cultured Retinal Pigment Epithelial Cells. Advances in experimental medicine and biology Calton, M. A., Vollrath, D. 2016; 854: 709-715


    Retinal pigment epithelium (RPE) cell migration in response to disease has been reported for age-related macular degeneration, proliferative vitreoretinopathy, and proliferative diabetic retinopathy. The complex molecular process of RPE cell migration is regulated in part by growth factors and cytokines, and activation of the PI3/AKT/mTOR signaling pathway. Rapamycin, an allosteric mTOR inhibitor, has been shown to block only one of the primary downstream mTOR effectors, p70 S6 kinase 1, in many cell types. INK128, a selective mTOR ATP binding site competitor, blocks both p70 S6 kinase 1 and a second primary downstream effector, 4E-BP1. We performed scratch assays using differentiated ARPE-19 and primary porcine RPE cells to assess the effect of mTOR inhibition on cell migration. We found that INK128-mediated blocking of both p70 S6 kinase 1 and 4E-BP1 was much more effective at preventing RPE cell migration than rapamycin-mediated inhibition of p70 S6 kinase 1 alone.

    View details for DOI 10.1007/978-3-319-17121-0_94

    View details for PubMedID 26427479

  • Tyro3 Modulates Mertk-Associated Retinal Degeneration. PLoS genetics Vollrath, D., Yasumura, D., Benchorin, G., Matthes, M. T., Feng, W., Nguyen, N. M., Sedano, C. D., Calton, M. A., LaVail, M. M. 2015; 11 (12): e1005723


    Inherited photoreceptor degenerations (IPDs) are the most genetically heterogeneous of Mendelian diseases. Many IPDs exhibit substantial phenotypic variability, but the basis is usually unknown. Mutations in MERTK cause recessive IPD phenotypes associated with the RP38 locus. We have identified a murine genetic modifier of Mertk-associated photoreceptor degeneration, the C57BL/6 (B6) allele of which acts as a suppressor. Photoreceptors degenerate rapidly in Mertk-deficient animals homozygous for the 129P2/Ola (129) modifier allele, whereas animals heterozygous for B6 and 129 modifier alleles exhibit an unusual intermixing of degenerating and preserved retinal regions, with females more severely affected than males. Mertk-deficient mice homozygous for the B6 modifier allele display degeneration only in the far periphery, even at 8 months of age, and have improved retinal function compared to animals homozygous for the 129 allele. We genetically mapped the modifier to an approximately 2-megabase critical interval that includes Tyro3, a paralog of Mertk. Tyro3 expression in the outer retina varies with modifier genotype in a manner characteristic of a cis-acting expression quantitative trait locus (eQTL), with the B6 allele conferring an approximately three-fold higher expression level. Loss of Tyro3 function accelerates the pace of photoreceptor degeneration in Mertk knockout mice, and TYRO3 protein is more abundant in the retinal pigment epithelium (RPE) adjacent to preserved central retinal regions of Mertk knockout mice homozygous for the B6 modifier allele. Endogenous human TYRO3 protein co-localizes with nascent photoreceptor outer segment (POS) phagosomes in a primary RPE cell culture assay, and expression of murine Tyro3 in cultured cells stimulates phagocytic ingestion of POS. Our findings demonstrate that Tyro3 gene dosage modulates Mertk-associated retinal degeneration, provide strong evidence for a direct role for TYRO3 in RPE phagocytosis, and suggest that an eQTL can modify a recessive IPD.

    View details for DOI 10.1371/journal.pgen.1005723

    View details for PubMedID 26656104

    View details for PubMedCentralID PMC4687644

  • SPP2 Mutations Cause Autosomal Dominant Retinitis Pigmentosa SCIENTIFIC REPORTS Liu, Y., Chen, X., Xu, Q., Gao, X., Tam, P. O., Zhao, K., Zhang, X., Chen, L. J., Jia, W., Zhao, Q., Vollrath, D., Pang, C. P., Zhao, C. 2015; 5


    Retinitis pigmentosa (RP) shows progressive loss of photoreceptors involved with heterogeneous genetic background. Here, by exome sequencing and linkage analysis on a Chinese family with autosomal dominant RP, we identified a putative pathogenic variant, p.Gly97Arg, in the gene SPP2, of which expression was detected in multiple tissues including retina. The p.Gly97Arg was absent in 800 ethnically matched chromosomes and 1400 in-house exome dataset, and was located in the first of the two highly conserved disulfide bonded loop of secreted phosphoprotein 2 (Spp-24) encoded by SPP2. Overexpression of p.Gly97Arg and another signal peptide mutation, p.Gly29Asp, caused cellular retention of both endogenous wild type and exogenous mutants in vitro, and primarily affected rod photoreceptors in zebrafish mimicking cardinal feature of RP. Taken together, our data indicate that the two mutations of SPP2 have dominant negative effects and cellular accumulation of Spp-24 might be particularly toxic to photoreceptors and/or retinal pigment epithelium. SPP2 has a new role in retinal degeneration.

    View details for DOI 10.1038/srep14867

    View details for Web of Science ID 000362641000002

    View details for PubMedID 26459573

    View details for PubMedCentralID PMC4602186

  • DNA copy number variants of known glaucoma genes in relation to primary open-angle glaucoma. Investigative ophthalmology & visual science Liu, Y., Garrett, M. E., Yaspan, B. L., Bailey, J. C., Loomis, S. J., Brilliant, M., Budenz, D. L., Christen, W. G., Fingert, J. H., Gaasterland, D., Gaasterland, T., Kang, J. H., Lee, R. K., Lichter, P., Moroi, S. E., Realini, A., Richards, J. E., Schuman, J. S., Scott, W. K., Singh, K., Sit, A. J., Vollrath, D., Weinreb, R., Wollstein, G., Zack, D. J., Zhang, K., Pericak-Vance, M. A., Haines, J. L., Pasquale, L. R., Wiggs, J. L., Allingham, R. R., Ashley-Koch, A. E., Hauser, M. A. 2014; 55 (12): 8251-8258


    We examined the role of DNA copy number variants (CNVs) of known glaucoma genes in relation to primary open angle glaucoma (POAG).Our study included DNA samples from two studies (NEIGHBOR and GLAUGEN). All the samples were genotyped with the Illumina Human660W_Quad_v1 BeadChip. After removing non-blood-derived and amplified DNA samples, we applied quality control steps based on the mean Log R Ratio and the mean B allele frequency. Subsequently, data from 3057 DNA samples (1599 cases and 1458 controls) were analyzed with PennCNV software. We defined CNVs as those ≥5 kilobases (kb) in size and interrogated by ≥5 consecutive probes. We further limited our investigation to CNVs in known POAG-related genes, including CDKN2B-AS1, TMCO1, SIX1/SIX6, CAV1/CAV2, the LRP12-ZFPM2 region, GAS7, ATOH7, FNDC3B, CYP1B1, MYOC, OPTN, WDR36, SRBD1, TBK1, and GALC.Genomic duplications of CDKN2B-AS1 and TMCO1 were each found in a single case. Two cases carried duplications in the GAS7 region. Genomic deletions of SIX6 and ATOH7 were each identified in one case. One case carried a TBK1 deletion and another case carried a TBK1 duplication. No controls had duplications or deletions in these six genes. A single control had a duplication in the MYOC region. Deletions of GALC were observed in five cases and two controls.The CNV analysis of a large set of cases and controls revealed the presence of rare CNVs in known POAG susceptibility genes. Our data suggest that these rare CNVs may contribute to POAG pathogenesis and merit functional evaluation.

    View details for DOI 10.1167/iovs.14-15712

    View details for PubMedID 25414181

  • Hypothesis-independent pathway analysis implicates GABA and Acetyl-CoA metabolism in primary open-angle glaucoma and normal-pressure glaucoma HUMAN GENETICS Bailey, J. N., Yaspan, B. L., Pasquale, L. R., Hauser, M. A., Kang, J. H., Loomis, S. J., Brilliant, M., Budenz, D. L., Christen, W. G., Fingert, J., Gaasterland, D., Gaasterland, T., Kraft, P., Lee, R. K., Lichter, P. R., Liu, Y., McCarty, C. A., Moroi, S. E., Richards, J. E., Realini, T., Schuman, J. S., Scott, W. K., Singh, K., Sit, A. J., Vollrath, D., Wollstein, G., Zack, D. J., Zhang, K., Pericak-Vance, M. A., Allingham, R. R., Weinreb, R. N., Haines, J. L., Wiggs, J. L. 2014; 133 (10): 1319-1330


    Primary open-angle glaucoma (POAG) is a leading cause of blindness worldwide. Using genome-wide association single-nucleotide polymorphism data from the Glaucoma Genes and Environment study and National Eye Institute Glaucoma Human Genetics Collaboration comprising 3,108 cases and 3,430 controls, we assessed biologic pathways as annotated in the KEGG database for association with risk of POAG. After correction for genic overlap among pathways, we found 4 pathways, butanoate metabolism (hsa00650), hematopoietic cell lineage (hsa04640), lysine degradation (hsa00310) and basal transcription factors (hsa03022) related to POAG with permuted p < 0.001. In addition, the human leukocyte antigen (HLA) gene family was significantly associated with POAG (p < 0.001). In the POAG subset with normal-pressure glaucoma (NPG), the butanoate metabolism pathway was also significantly associated (p < 0.001) as well as the MAPK and Hedgehog signaling pathways (hsa04010 and hsa04340), glycosaminoglycan biosynthesis-heparan sulfate pathway (hsa00534) and the phenylalanine, tyrosine and tryptophan biosynthesis pathway (hsa0400). The butanoate metabolism pathway overall, and specifically the aspects of the pathway that contribute to GABA and acetyl-CoA metabolism, was the only pathway significantly associated with both POAG and NPG. Collectively these results implicate GABA and acetyl-CoA metabolism in glaucoma pathogenesis, and suggest new potential therapeutic targets.

    View details for DOI 10.1007/s00439-014-1468-7

    View details for Web of Science ID 000341828900009

    View details for PubMedCentralID PMC4273559

  • Intrastriatal Transplantation of Retinal Pigment Epithelial Cells for the Treatment of Parkinson Disease: In Vivo Longitudinal Molecular Imaging with (18)F-P3BZA PET/CT. Radiology Bu, L., Li, R., Liu, H., Feng, W., Xiong, X., Zhao, H., Vollrath, D., Shen, B., Cheng, Z. 2014; 272 (1): 174-183


    Purpose To evaluate the performance of N-[2-(diethylamino)ethyl]-(18)F-5-fluoropicolinamide ((18)F-P3BZA) for visualizing porcine retinal pigment epithelium (pRPE) cells transplanted in the striatum for the treatment of Parkinson disease and to monitor the long-term activity of implanted pRPE cells by means of (18)F-P3BZA positron emission tomography (PET)/computed tomography (CT) in vivo. Materials and Methods Animal work was conducted in accordance with the administrative panel on laboratory animal care. In vitro cell uptake of (18)F-P3BZA was determined with incubation of melanotic pRPE or amelanotic ARPE-19 cells with (18)F-P3BZA. To visualize the implanted pRPE cells in vivo, normal rats (four per group) were injected with pRPE or ARPE-19 cells attached to gelatin microcarriers in the left striatum and with control gelatin microcarriers in the right striatum and followed up with small animal PET/CT. Longitudinal PET/CT scans were acquired in 12 rats up to 16 days after surgery. Postmortem analysis, which included autoradiography and hematoxylin-eosin, Fontana-Masson, and immunofluorescence staining, was performed. Data were compared with the Student t test, analysis of variance, and regression analysis. Results (18)F-P3BZA accumulated in pRPE cells effectively (3.48% of the injected dose [ID] per gram of brain tissue ± 0.58 at 1 hour after injection of the probe at 2 days after surgery in vivo) but not in control ARPE-19 cells (P < .05). Longitudinal PET/CT scans revealed that the activity of implanted pRPE cells decreased over time, as evidenced by a reduction in (18)F-P3BZA uptake (3.39% ID/g ± 0.18, 2.49% ID/g ± 0.41, and 1.20% ID/g ± 0.13 at days 2, 9, and 16, respectively; P < .05). Postmortem analysis helped confirm the results of in vivo imaging. Conclusion (18)F-P3BZA PET/CT is a feasible technique for visualizing and detecting the activity of implanted RPE cells in vivo. © RSNA, 2014 Online supplemental material is available for this article.

    View details for DOI 10.1148/radiol.14132042

    View details for PubMedID 24758555

  • PRPF4 mutations cause autosomal dominant retinitis pigmentosa HUMAN MOLECULAR GENETICS Chen, X., Liu, Y., Sheng, X., Tam, P. O., Zhao, K., Chen, X., Rong, W., Liu, Y., Liu, X., Pan, X., Chen, L. J., Zhao, Q., Vollrath, D., Pang, C. P., Zhao, C. 2014; 23 (11): 2926-2939


    Retinitis pigmentosa (RP), a disease characterized by progressive loss of photoreceptors, exhibits significant genetic heterogeneity. Several genes associated with U4/U6-U5 triple small nuclear ribonucleoprotein (tri-snRNP) complex of the spliceosome have been implicated in autosomal dominant RP (adRP). HPrp4, encoded by PRPF4, regulates the stability of U4/U6 di-snRNP, which is essential for continuous splicing. Here, we identified two heterozygous variants in PRPF4, including c.-114_-97del in a simplex RP patient and c.C944T (p.Pro315Leu), which co-segregates with disease phenotype in a family with adRP. Both variants were absent in 400 unrelated controls. The c.-114_-97del, predicted to affect two transcription factor binding sites, was shown to down-regulate the promoter activity of PRPF4 by a luciferase assay, and was associated with a significant reduction of PRPF4 expression in the blood cells of the patient. In fibroblasts from an affected individual with the p.Pro315Leu variant, the expression levels of several tri-snRNP components, including PRPF4 itself, were up-regulated, with altered expression pattern of SC35, a spliceosome marker. The same alterations were also observed in cells over expressing hPrp4(Pro315Leu), suggesting that they arose as a compensatory response to a compromised splicing mechanism caused by hPrp4 dysfunction. Further, over expression of hPrp4(Pro315Leu), but not hPrp4(WT), triggered systemic deformities in wild-type zebrafish embryos with the retina primarily affected, and dramatically augmented death rates in morphant embryos, in which orthologous zebrafish prpf4 gene was silenced. We conclude that mutations of PRPF4 cause RP via haploinsufficiency and dominant-negative effects, and establish PRPF4 as a new U4/U6-U5 snRNP component associated with adRP.

    View details for DOI 10.1093/hmg/ddu005

    View details for Web of Science ID 000336483200011

  • Vascular tone pathway polymorphisms in relation to primary open-angle glaucoma EYE Kang, J. H., Loomis, S. J., Yaspan, B. L., BAILEY, J. C., Weinreb, R. N., Lee, R. K., Lichter, P. R., Budenz, D. L., Liu, Y., Realini, T., Gaasterland, D., Gaasterland, T., Friedman, D. S., McCarty, C. A., Moroi, S. E., Olson, L., Schuman, J. S., Singh, K., Vollrath, D., Wollstein, G., Zack, D. J., Brilliant, M., Sit, A. J., Christen, W. G., Fingert, J., Forman, J. P., Buys, E. S., Kraft, P., Zhang, K., Allingham, R. R., Pericak-Vance, M. A., Richards, J. E., Hauser, M. A., Haines, J. L., Wiggs, J. L., Pasquale, L. R. 2014; 28 (6): 662-671


    AimsVascular perfusion may be impaired in primary open-angle glaucoma (POAG); thus, we evaluated a panel of markers in vascular tone-regulating genes in relation to POAG.MethodsWe used Illumina 660W-Quad array genotype data and pooled P-values from 3108 POAG cases and 3430 controls from the combined National Eye Institute Glaucoma Human Genetics Collaboration consortium and Glaucoma Genes and Environment studies. Using information from previous literature and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, we compiled single-nucleotide polymorphisms (SNPs) in 186 vascular tone-regulating genes. We used the 'Pathway Analysis by Randomization Incorporating Structure' analysis software, which performed 1000 permutations to compare the overall pathway and selected genes with comparable randomly generated pathways and genes in their association with POAG.ResultsThe vascular tone pathway was not associated with POAG overall or POAG subtypes, defined by the type of visual field loss (early paracentral loss (n=224 cases) or only peripheral loss (n=993 cases)) (permuted P≥0.20). In gene-based analyses, eight were associated with POAG overall at permuted P<0.001: PRKAA1, CAV1, ITPR3, EDNRB, GNB2, DNM2, HFE, and MYL9. Notably, six of these eight (the first six listed) code for factors involved in the endothelial nitric oxide synthase activity, and three of these six (CAV1, ITPR3, and EDNRB) were also associated with early paracentral loss at P<0.001, whereas none of the six genes reached P<0.001 for peripheral loss only.DiscussionAlthough the assembled vascular tone SNP set was not associated with POAG, genes that code for local factors involved in setting vascular tone were associated with POAG.

    View details for DOI 10.1038/eye.2014.42

    View details for Web of Science ID 000338340200004

    View details for PubMedCentralID PMC4058608

  • Association of CAV1/CAV2 genomic variants with primary open-angle glaucoma overall and by gender and pattern of visual field loss. Ophthalmology Loomis, S. J., Kang, J. H., Weinreb, R. N., Yaspan, B. L., Cooke Bailey, J. N., Gaasterland, D., Gaasterland, T., Lee, R. K., Lichter, P. R., Budenz, D. L., Liu, Y., Realini, T., Friedman, D. S., McCarty, C. A., Moroi, S. E., Olson, L., Schuman, J. S., Singh, K., Vollrath, D., Wollstein, G., Zack, D. J., Brilliant, M., Sit, A. J., Christen, W. G., Fingert, J., Kraft, P., Zhang, K., Allingham, R. R., Pericak-Vance, M. A., Richards, J. E., Hauser, M. A., Haines, J. L., Pasquale, L. R., Wiggs, J. L. 2014; 121 (2): 508-516


    The CAV1/CAV2 (caveolin 1 and caveolin 2) genomic region previously was associated with primary open-angle glaucoma (POAG), although replication among independent studies has been variable. The aim of this study was to assess the association between CAV1/CAV2 single nucleotide polymorphisms (SNPs) and POAG in a large case-control dataset and to explore associations by gender and pattern of visual field (VF) loss further.Case-control study.We analyzed 2 large POAG data sets: the Glaucoma Genes and Environment (GLAUGEN) study (976 cases, 1140 controls) and the National Eye Institute Glaucoma Human Genetics Collaboration (NEIGHBOR) consortium (2132 cases, 2290 controls).We studied the association between 70 SNPs located within the CAV1/CAV2 genomic region in the GLAUGEN and NEIGHBOR studies, both genotyped on the Illumina Human 660WQuadv1C BeadChip array and imputed with the Markov Chain Haplotyping algorithm using the HapMap 3 reference panel. We used logistic regression models of POAG in the overall population and separated by gender, as well as by POAG subtypes defined by type of VF defect (peripheral or paracentral). Results from GLAUGEN and NEIGHBOR were meta-analyzed, and a Bonferroni-corrected significance level of 7.7 × 10(-4) was used to account for multiple comparisons.Overall POAG, overall POAG by gender, and POAG subtypes defined by pattern of early VF loss.We found significant associations between 10 CAV1/CAV2 SNPs and POAG (top SNP, rs4236601; pooled P = 2.61 × 10(-7)). Of these, 9 were significant only in women (top SNP, rs4236601; pooled P = 1.59 × 10(-5)). Five of the 10 CAV1/CAV2 SNPs were associated with POAG with early paracentral VF (top SNP, rs17588172; pooled P = 1.07 × 10(-4)), and none of the 10 were associated with POAG with peripheral VF loss only or POAG among men.CAV1/CAV2 SNPs were associated significantly with POAG overall, particularly among women. Furthermore, we found an association between CAV1/CAV2 SNPs and POAG with paracentral VF defects. These data support a role for caveolin 1, caveolin 2, or both in POAG and suggest that the caveolins particularly may affect POAG pathogenesis in women and in patients with early paracentral VF defects.

    View details for DOI 10.1016/j.ophtha.2013.09.012

    View details for PubMedID 24572674

  • Estrogen pathway polymorphisms in relation to primary open angle glaucoma: An analysis accounting for gender from the United States MOLECULAR VISION Pasquale, L. R., Loomis, S. J., Weinreb, R. N., Kang, J. H., Yaspan, B. L., Bailey, J. C., Gaasterland, D., Gaasterland, T., Lee, R. K., Scott, W. K., Lichter, P. R., Budenz, D. L., Liu, Y., Realini, T., Friedman, D. S., McCarty, C. A., Moroi, S. E., Olson, L., Schuman, J. S., Singh, K., Vollrath, D., Wollstein, G., Zack, D. J., Brilliant, M., Sit, A. J., Christen, W. G., Fingert, J., Kraft, P., Zhang, K., Allingham, R. R., Pericak-Vance, M. A., Richards, J. E., Hauser, M. A., Haines, J. L., Wiggs, J. L. 2013; 19: 1471-1481


    Circulating estrogen levels are relevant in glaucoma phenotypic traits. We assessed the association between an estrogen metabolism single nucleotide polymorphism (SNP) panel in relation to primary open angle glaucoma (POAG), accounting for gender.We included 3,108 POAG cases and 3,430 controls of both genders from the Glaucoma Genes and Environment (GLAUGEN) study and the National Eye Institute Glaucoma Human Genetics Collaboration (NEIGHBOR) consortium genotyped on the Illumina 660W-Quad platform. We assessed the relation between the SNP panels representative of estrogen metabolism and POAG using pathway- and gene-based approaches with the Pathway Analysis by Randomization Incorporating Structure (PARIS) software. PARIS executes a permutation algorithm to assess statistical significance relative to the pathways and genes of comparable genetic architecture. These analyses were performed using the meta-analyzed results from the GLAUGEN and NEIGHBOR data sets. We evaluated POAG overall as well as two subtypes of POAG defined as intraocular pressure (IOP) ≥22 mmHg (high-pressure glaucoma [HPG]) or IOP <22 mmHg (normal pressure glaucoma [NPG]) at diagnosis. We conducted these analyses for each gender separately and then jointly in men and women.Among women, the estrogen SNP pathway was associated with POAG overall (permuted p=0.006) and HPG (permuted p<0.001) but not NPG (permuted p=0.09). Interestingly, there was no relation between the estrogen SNP pathway and POAG when men were considered alone (permuted p>0.99). Among women, gene-based analyses revealed that the catechol-O-methyltransferase gene showed strong associations with HTG (permuted gene p≤0.001) and NPG (permuted gene p=0.01).The estrogen SNP pathway was associated with POAG among women.

    View details for Web of Science ID 000321786700003

    View details for PubMedID 23869166

    View details for PubMedCentralID PMC3712669

  • A Novel Homozygous BEST1 Mutation Correlates with Complex Ocular Phenotypes OPHTHALMOLOGY Sheng, X., Chen, X., Zhao, K., Liu, Y., Vollrath, D., Zhao, C. 2013; 120 (7): 1511-+

    View details for DOI 10.1016/j.ophtha.2013.03.043

    View details for Web of Science ID 000321108500054

    View details for PubMedID 23823511

  • Targeted Sequencing of 179 Genes Associated with Hereditary Retinal Dystrophies and 10 Candidate Genes Identifies Novel and Known Mutations in Patients with Various Retinal Diseases INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE Chen, X., Zhao, K., Sheng, X., Li, Y., Gao, X., Zhang, X., Kang, X., Pan, X., Liu, Y., Jiang, C., Shi, H., Chen, X., Rong, W., Chen, L. J., Lai, T. Y., Liu, Y., Wang, X., Yuan, S., Liu, Q., Vollrath, D., Pang, C. P., Zhao, C. 2013; 54 (3): 2186-2197


    Hereditary retinal dystrophies (HRDs) are a group of monogenic diseases characterized by an irreversible loss of photoreceptors. HRDs exhibit significant genetic and clinical heterogeneities challenging traditional techniques for determining disease-causal mutations. This study aims to develop an efficient molecular diagnostic platform for HRDs, and to determine the genetic basis for 25 randomly collected Chinese families with a variety of HRDs.We designed a high throughput sequence capture microarray targeting 179 genes associated with HRDs and 10 candidate genes. We combined sequence capture with next-generation sequencing (NGS) to screen for mutations in the cohort of Chinese families. Variants detected by NGS were filtered, validated, and prioritized by pathogenicity analysis. Genotypes and phenotypes were correlated.We identified four recurrent single mutations, two compound mutations, and eight novel putative causative mutations, including five putative pathogenic alleles (e.g., premature stop codons and frame shifts) and three novel missense variants that are very likely pathogenic. These findings provided specific genetic diagnoses in 14 of 25 families (56%). Among these, identification of a mutation in VCAN in a family with a complicated phenotype helped to finalize the clinical diagnosis as Wagner syndrome. In another five families, 11 potential novel pathogenic variants were identified.A substantial number of potential new genes and new mutations associated with HRDs remain to be discovered. Identification of the novel HRDs-causing mutations in our study not only provides a better understanding of genotype-phenotype relationships in these diseases, but also demonstrates that the approach described herein is an effective method for large scale mutation detection among diverse and complicated HRDs cases.

    View details for DOI 10.1167/iovs.12-10967

    View details for Web of Science ID 000316942400081

    View details for PubMedID 23462753

  • CDKN2B-AS1 Genotype-Glaucoma Feature Correlations in Primary Open-Angle Glaucoma Patients From the United States AMERICAN JOURNAL OF OPHTHALMOLOGY Pasquale, L. R., Loomis, S. J., Kang, J. H., Yaspan, B. L., Abdrabou, W., Budenz, D. L., Chen, T. C., DelBono, E., Friedman, D. S., Gaasterland, D., Gaasterland, T., Grosskreutz, C. L., Lee, R. K., Lichter, P. R., Liu, Y., McCarty, C. A., Moroi, S. E., Olson, L. M., Realini, T., Rhee, D. J., Schuman, J. S., Singh, K., Vollrath, D., Wollstein, G., Zack, D. J., Allingham, R. R., Pericak-Vance, M. A., Weinreb, R. N., Zhang, K., Hauser, M. A., Richards, J. E., Haines, J. L., Wiggs, J. L. 2013; 155 (2): 342-353


    To assess the association between single nucleotide polymorphisms (SNPs) of the gene region containing cyclin-dependent kinase inhibitor 2B antisense noncoding RNA (CDKN2B-AS1) and glaucoma features among primary open-angle glaucoma (POAG) patients.Retrospective observational case series.We studied associations between 10 CDKN2B-AS1 SNPs and glaucoma features among 976 POAG cases from the Glaucoma Genes and Environment (GLAUGEN) study and 1971 cases from the National Eye Institute Glaucoma Human Genetics Collaboration (NEIGHBOR) consortium. For each patient, we chose the feature from the eye with the higher value. We created cohort-specific multivariable models for glaucoma features and then meta-analyzed the results.For 9 of the 10 protective CDKN2B-AS1 SNPs with minor alleles associated with reduced disease risk (eg, the G allele at rs2157719), POAG patients carrying these minor alleles had smaller cup-to-disc ratio (0.05 units smaller per G allele at diagnosis; 95% CI: -0.08, -0.03; P = 6.23E-05) despite having higher intraocular pressure (IOP) (0.70 mm Hg higher per G allele at DNA collection; 95% CI: 0.40, 1.00; P = 5.45E-06). For the 1 adverse rs3217992 SNP with minor allele A associated with increased disease risk, POAG patients with A alleles had larger cup-to-disc ratio (0.05 units larger per A allele at diagnosis; 95% CI: 0.02, 0.07; P = 4.74E-04) despite having lower IOP (-0.57 mm Hg per A allele at DNA collection; 95% CI: -0.84, -0.29; P = 6.55E-05).Alleles of CDKN2B-AS1 SNPs, which influence risk of developing POAG, also modulate optic nerve degeneration among POAG patients, underscoring the role of CDKN2B-AS1 in POAG.

    View details for DOI 10.1016/j.ajo.2012.07.023

    View details for Web of Science ID 000314137400019

    View details for PubMedID 23111177

    View details for PubMedCentralID PMC3544983

  • Amyloid Fibril Formation by the Glaucoma-Associated Olfactomedin Domain of Myocilin JOURNAL OF MOLECULAR BIOLOGY Orwig, S. D., Perry, C. W., Kim, L. Y., Turnage, K. C., Zhang, R., Vollrath, D., Schmidt-Krey, I., Lieberman, R. L. 2012; 421 (2-3): 242-255


    Myocilin is a protein found in the extracellular matrix of trabecular meshwork tissue, the anatomical region of the eye involved in regulating intraocular pressure. Wild-type (WT) myocilin has been associated with steroid-induced glaucoma, and variants of myocilin have been linked to early-onset inherited glaucoma. Elevated levels and aggregation of myocilin hasten increased intraocular pressure and glaucoma-characteristic vision loss due to irreversible damage to the optic nerve. In spite of reports on the intracellular accumulation of mutant and WT myocilin in vitro, cell culture, and model organisms, these aggregates have not been structurally characterized. In this work, we provide biophysical evidence for the hallmarks of amyloid fibrils in aggregated forms of WT and mutant myocilin localized to the C-terminal olfactomedin (OLF) domain. These fibrils are grown under a variety of conditions in a nucleation-dependent and self-propagating manner. Protofibrillar oligomers and mature amyloid fibrils are observed in vitro. Full-length mutant myocilin expressed in mammalian cells forms intracellular amyloid-containing aggregates as well. Taken together, this work provides new insights into and raises new questions about the molecular properties of the highly conserved OLF domain, and suggests a novel protein-based hypothesis for glaucoma pathogenesis for further testing in a clinical setting.

    View details for DOI 10.1016/j.jmb.2011.12.016

    View details for Web of Science ID 000306885200008

    View details for PubMedID 22197377

    View details for PubMedCentralID PMC3323732

  • Genome-Wide Analysis of Central Corneal Thickness in Primary Open-Angle Glaucoma Cases in the NEIGHBOR and GLAUGEN Consortia INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE Ulmer, M., Li, J., Yaspan, B. L., Ozel, A. B., Richards, J. E., Moroi, S. E., Hawthorne, F., Budenz, D. L., Friedman, D. S., Gaasterland, D., Haines, J., Kang, J. H., Lee, R., Lichter, P., Liu, Y., Pasquale, L. R., Pericak-Vance, M., Realini, A., Schuman, J. S., Singh, K., Vollrath, D., Weinreb, R., Wollstein, G., Zack, D. J., Zhang, K., Young, T., Allingham, R. R., Wiggs, J. L., Ashley-Koch, A., Hauser, M. A. 2012; 53 (8): 4468-4474


    To investigate the effects of central corneal thickness (CCT)-associated variants on primary open-angle glaucoma (POAG) risk using single nucleotide polymorphisms (SNP) data from the Glaucoma Genes and Environment (GLAUGEN) and National Eye Institute (NEI) Glaucoma Human Genetics Collaboration (NEIGHBOR) consortia.A replication analysis of previously reported CCT SNPs was performed in a CCT dataset (n = 1117) and these SNPs were then tested for association with POAG using a larger POAG dataset (n = 6470). Then a CCT genome-wide association study (GWAS) was performed. Top SNPs from this analysis were selected and tested for association with POAG. cDNA libraries from fetal and adult brain and ocular tissue samples were generated and used for candidate gene expression analysis.Association with one of 20 previously published CCT SNPs was replicated: rs12447690, near the ZNF469 gene (P = 0.001; β = -5.08 μm/allele). None of these SNPs were significantly associated with POAG. In the CCT GWAS, no SNPs reached genome-wide significance. After testing 50 candidate SNPs for association with POAG, one SNP was identified, rs7481514 within the neurotrimin (NTM) gene, that was significantly associated with POAG in a low-tension subset (P = 0.00099; Odds Ratio [OR] = 1.28). Additionally, SNPs in the CNTNAP4 gene showed suggestive association with POAG (top SNP = rs1428758; P = 0.018; OR = 0.84). NTM and CNTNAP4 were shown to be expressed in ocular tissues.The results suggest previously reported CCT loci are not significantly associated with POAG susceptibility. By performing a quantitative analysis of CCT and a subsequent analysis of POAG, SNPs in two cell adhesion molecules, NTM and CNTNAP4, were identified and may increase POAG susceptibility in a subset of cases.

    View details for DOI 10.1167/iovs.12-9784

    View details for Web of Science ID 000307096400018

    View details for PubMedID 22661486

    View details for PubMedCentralID PMC3394688

  • Tyrosine-Mutant AAV8 Delivery of Human MERTK Provides Long-Term Retinal Preservation in RCS Rats INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE Deng, W., Dinculescu, A., Li, Q., Boye, S. L., Li, J., Gorbatyuk, M. S., Pang, J., Chiodo, V. A., Matthes, M. T., Yasumura, D., Liu, L., Alkuraya, F. S., Zhang, K., Vollrath, D., LaVail, M. M., Hauswirth, W. W. 2012; 53 (4): 1895-1904


    The absence of Mertk in RCS rats results in defective RPE phagocytosis, accumulation of outer segment (OS) debris in the subretinal space, and subsequent death of photoreceptors. Previous research utilizing Mertk gene replacement therapy in RCS rats provided proof of concept for treatment of this form of recessive retinitis pigmentosa (RP); however, the beneficial effects on retinal function were transient. In the present study, we evaluated whether delivery of a MERTK transgene using a tyrosine-mutant AAV8 capsid could lead to more robust and longer-term therapeutic outcomes than previously reported.An AAV8 Y733F vector expressing a human MERTK cDNA driven by a RPE-selective promoter was administrated subretinally at postnatal day 2. Functional and morphological analyses were performed at 4 months and 8 months post-treatment. Retinal vasculature and Müller cell activation were analyzed by quantifying acellular capillaries and glial fibrillary acidic protein immunostaining, respectively.Electroretinographic responses from treated eyes were more than one-third of wild-type levels and OS were well preserved in the injection area even at 8 months. Rescue of RPE phagocytosis, prevention of retinal vasculature degeneration, and inhibition of Müller cell activation were demonstrated in the treated eyes for at least 8 months.This research describes a longer and much more robust functional and morphological rescue than previous studies. We also demonstrate for the first time that an AAV8 mutant capsid serotype vector has a substantial therapeutic potential for RPE-specific gene delivery. These results suggest that tyrosine-mutant AAV8 vectors hold promise for the treatment of individuals with MERTK-associated RP.

    View details for DOI 10.1167/iovs.11-8831

    View details for Web of Science ID 000303669400025

    View details for PubMedID 22408006

    View details for PubMedCentralID PMC3995567

  • Common Variants at 9p21 and 8q22 Are Associated with Increased Susceptibility to Optic Nerve Degeneration in Glaucoma PLOS GENETICS Wiggs, J. L., Yapan, B. L., Hauser, M. A., Kane, J. H., Allingham, R. R., Olson, L. M., Abdrabou, W., Fan, B. J., Wang, D. Y., Brodeur, W., Budenz, D. L., Caprioli, J., Crenshaw, A., Crooks, K., DelBono, E., Doheny, K. F., Friedman, D. S., Gaasterland, D., Gaasterland, T., Laurie, C., Lee, R. K., Lichter, P. R., Loomis, S., Liu, Y., Medeiros, F. A., McCarty, C., Mirel, D., Moroi, S. E., Musch, D. C., Realini, A., Rozsa, F. W., Schuman, J. S., Scott, K., Singh, K., Stein, J. D., Trager, E. H., VanVeldhuisen, P., Vollrath, D., Wollstein, G., Yoneyama, S., Zhang, K., Weinreb, R. N., Ernst, J., Kellis, M., Masuda, T., Zack, D., Richards, J. E., Pericak-Vance, M., Pasquale, L. R., Haines, J. L. 2012; 8 (4): 413-424


    Optic nerve degeneration caused by glaucoma is a leading cause of blindness worldwide. Patients affected by the normal-pressure form of glaucoma are more likely to harbor risk alleles for glaucoma-related optic nerve disease. We have performed a meta-analysis of two independent genome-wide association studies for primary open angle glaucoma (POAG) followed by a normal-pressure glaucoma (NPG, defined by intraocular pressure (IOP) less than 22 mmHg) subgroup analysis. The single-nucleotide polymorphisms that showed the most significant associations were tested for association with a second form of glaucoma, exfoliation-syndrome glaucoma. The overall meta-analysis of the GLAUGEN and NEIGHBOR dataset results (3,146 cases and 3,487 controls) identified significant associations between two loci and POAG: the CDKN2BAS region on 9p21 (rs2157719 [G], OR = 0.69 [95%CI 0.63-0.75], p = 1.86×10⁻¹⁸), and the SIX1/SIX6 region on chromosome 14q23 (rs10483727 [A], OR = 1.32 [95%CI 1.21-1.43], p = 3.87×10⁻¹¹). In sub-group analysis two loci were significantly associated with NPG: 9p21 containing the CDKN2BAS gene (rs2157719 [G], OR = 0.58 [95% CI 0.50-0.67], p = 1.17×10⁻¹²) and a probable regulatory region on 8q22 (rs284489 [G], OR = 0.62 [95% CI 0.53-0.72], p = 8.88×10⁻¹⁰). Both NPG loci were also nominally associated with a second type of glaucoma, exfoliation syndrome glaucoma (rs2157719 [G], OR = 0.59 [95% CI 0.41-0.87], p = 0.004 and rs284489 [G], OR = 0.76 [95% CI 0.54-1.06], p = 0.021), suggesting that these loci might contribute more generally to optic nerve degeneration in glaucoma. Because both loci influence transforming growth factor beta (TGF-beta) signaling, we performed a genomic pathway analysis that showed an association between the TGF-beta pathway and NPG (permuted p = 0.009). These results suggest that neuro-protective therapies targeting TGF-beta signaling could be effective for multiple forms of glaucoma.

    View details for DOI 10.1371/journal.pgen.1002654

    View details for Web of Science ID 000303441800031

    View details for PubMedID 22570617

    View details for PubMedCentralID PMC3343074

  • An ENU-Induced Mutation in the Mertk Gene (Mertk(nmf12)) Leads to a Slow Form of Retinal Degeneration INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE Maddox, D. M., Hicks, W. L., Vollrath, D., LaVail, M. M., Naggert, J. K., Nishina, P. M. 2011; 52 (7): 4703-4709


    To determine the basis and to characterize the phenotype of a chemically induced mutation in a mouse model of retinal degeneration.Screening by indirect ophthalmoscopy identified a line of N-ethyl-N-nitrosourea (ENU) mutagenized mice demonstrating retinal patches. Longitudinal studies of retinal histologic sections showed photoreceptors in the peripheral retina undergoing slow, progressive degeneration. The mutation was named neuroscience mutagenesis facility 12 (nmf12), and mapping localized the critical region to Chromosome 2.Sequencing of nmf12 DNA revealed a point mutation in the c-mer tyrosine kinase gene, designated Mertk(nmf12). We detected elevated levels of tumor necrosis factor (Tnf, previously Tnfa) in retinas of Mertk(nmf12) homozygotes relative to wild-type controls and investigated whether the increase of TNF, an inflammatory cytokine produced by macrophages/monocytes that signals intracellularly to cause necrosis or apoptosis, could underlie the retinal degeneration observed in Mertk(nmf12) homozygotes. Mertk(nmf12) homozygous mice were mated to mice lacking the entire Tnf gene and partial coding sequences of the Lta (Tnfb) and Ltb (Tnfc) genes.(2) B6.129P2-Ltb/Tnf/Lta(tm1Dvk)/J homozygotes did not exhibit a retinal degeneration phenotype and will, hereafter, be referred to as Tnfabc(-/-) mice. Surprisingly, mice homozygous for both the Mertk(nmf12) and the Ltb/Tnf/Lta(tm1Dvk) allele (Tnfabc(-/-)) demonstrated an increase in the rate of retinal degeneration.These findings illustrate that a mutation in the Mertk gene leads to a significantly slower progressive retinal degeneration compared with other alleles of Mertk. These results demonstrate that TNF family members play a role in protecting photoreceptors of Mertk(nmf12) homozygotes from cell death.

    View details for DOI 10.1167/iovs.10-7077

    View details for Web of Science ID 000293332500103

    View details for PubMedID 21436282

    View details for PubMedCentralID PMC3175976

  • mTOR pathway activation in age-related retinal disease AGING-US Zhao, C., Vollrath, D. 2011; 3 (4): 346-347

    View details for Web of Science ID 000290230800007

    View details for PubMedID 21483039

    View details for PubMedCentralID PMC3117448

  • Generation of Cre Transgenic Mice with Postnatal RPE-Specific Ocular Expression INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE Iacovelli, J., Zhao, C., Wolkow, N., Veldman, P., Gollomp, K., Ojha, P., Lukinova, N., King, A., Feiner, L., Esumi, N., Zack, D. J., Pierce, E. A., Vollrath, D., Dunaief, J. L. 2011; 52 (3): 1378-1383


    To generate and characterize a constitutively active, RPE-specific, cre-expressing transgenic mouse line. This line can be used to create RPE-specific knockouts by crossing with mice harboring loxP-flanked (floxed) genes.A transgene construct was assembled with the BEST1 promoter driving cre expression. Transgenic mice were generated on a C57BL/6 background. Cre expression was assessed by immunofluorescence and Western blot analysis. Cre enzymatic activity was tested by crossing to three lines with floxed DNA regions and detecting deletion of the intervening sequences or through histochemical detection of lacZ activity. Potential cre-mediated toxicity was assessed by retinal histology up to 24 months of age and by electroretinography.The BEST1-cre line with expression in the highest percentage of RPE cells displayed a patchy mosaic expression pattern, with 50% to 90% of RPE cells expressing cre. In mice outcrossed to a mixed B6/129 background, expression was consistently found in 90% of RPE cells. Within the eye, only the RPE cells were immunoreactive with an anti-cre antibody. Maximum cre expression quantified by Western blot analysis occurred at P28. Crosses with three lines containing floxed sequences revealed RPE-specific cre activity in the eye and extraocular expression limited to the testes. Histology and electroretinography showed no cre-mediated RPE toxicity.This BEST1-cre transgenic line enables generation of RPE-specific knockout mice. The mosaic expression pattern provides an internal control; the non-cre-expressing RPE cells continue to express the floxed genes. These mice should facilitate study of the multifunctional RPE and the generation of mouse models of human retinal disease.

    View details for DOI 10.1167/iovs.10-6347

    View details for Web of Science ID 000288965300023

    View details for PubMedID 21212186

    View details for PubMedCentralID PMC3101664

  • mTOR-mediated dedifferentiation of the retinal pigment epithelium initiates photoreceptor degeneration in mice JOURNAL OF CLINICAL INVESTIGATION Zhao, C., Yasumura, D., Li, X., Matthes, M., Lloyd, M., Nielsen, G., Ahern, K., Snyder, M., Bok, D., Dunaief, J. L., LaVail, M. M., Vollrath, D. 2011; 121 (1): 369-383


    Retinal pigment epithelial (RPE) cell dysfunction plays a central role in various retinal degenerative diseases, but knowledge is limited regarding the pathways responsible for adult RPE stress responses in vivo. RPE mitochondrial dysfunction has been implicated in the pathogenesis of several forms of retinal degeneration. Here we have shown that postnatal ablation of RPE mitochondrial oxidative phosphorylation in mice triggers gradual epithelium dedifferentiation, typified by reduction of RPE-characteristic proteins and cellular hypertrophy. The electrical response of the retina to light decreased and photoreceptors eventually degenerated. Abnormal RPE cell behavior was associated with increased glycolysis and activation of, and dependence upon, the hepatocyte growth factor/met proto-oncogene pathway. RPE dedifferentiation and hypertrophy arose through stimulation of the AKT/mammalian target of rapamycin (AKT/mTOR) pathway. Administration of an oxidant to wild-type mice also caused RPE dedifferentiation and mTOR activation. Importantly, treatment with the mTOR inhibitor rapamycin blunted key aspects of dedifferentiation and preserved photoreceptor function for both insults. These results reveal an in vivo response of the mature RPE to diverse stressors that prolongs RPE cell survival at the expense of epithelial attributes and photoreceptor function. Our findings provide a rationale for mTOR pathway inhibition as a therapeutic strategy for retinal degenerative diseases involving RPE stress.

    View details for DOI 10.1172/JCI44303

    View details for Web of Science ID 000285892300039

    View details for PubMedID 21135502

    View details for PubMedCentralID PMC3007156

  • Focus on Molecules: MERTK EXPERIMENTAL EYE RESEARCH Strick, D. J., Vollrath, D. 2010; 91 (6): 786-787

    View details for DOI 10.1016/j.exer.2010.05.006

    View details for Web of Science ID 000285218500001

    View details for PubMedID 20488176

    View details for PubMedCentralID PMC3133776

  • Candidate genes for chromosomes 6 and 10 quantitative trait loci for age-related retinal degeneration in mice MOLECULAR VISION Ogando, D. G., Dahlquist, K. D., Alizadeh, M., Kunchithapautham, K., Li, J., Yu, N., LaVail, M. M., Rohrer, B., Vollrath, D., Danciger, M. 2010; 16 (111-13): 1004-1018


    In a previous study, several quantitative trait loci (QTL) that influence age-related degeneration (ageRD) were identified in a cross between the albino strains B6(Cg)-Tyr(c-2J)/J (B6a) and BALB/cByJ (C). The Chromosome (Chr) 6 and Chr 10 QTL were the strongest and most highly significant loci and both involved B6a protective alleles. The QTL were responsible for 21% and 9% of the variance in phenotypes, respectively. We focused on these two QTL to identify candidate genes.DNA microarrays were used for the two mouse strains at four and eight months of age to identify genes that are differentially regulated and map to either QTL. Gene Ontology (GO) analysis of the differentially expressed genes was performed to identify possible processes and pathways associated with ageRD. To identify additional candidates, database analyses (Positional Medline or PosMed) were used. Based on differential expression, PosMed, and the presence of reported polymorphisms, five genes per QTL were selected for further study by sequencing analysis and qRT-PCR. Tumor necrosis factor, alpha- induced protein 3 (Tnfaip3; on a C57BL/6J (B6) background) was phenotypically tested. Single nucleotide polymorphisms (SNPs) flanking this gene were correlated with outer nuclear layer thickness (ONL), and eight-month-old Tnfaip3(+/-) mice were tested for ageRD.Polymorphisms were found in the coding regions of eight genes. Changes in gene expression were identified by qRT-PCR for Hexokinase 2 (Hk2) and Docking protein 1 (Dok1) at four months and for Dok1 and Tnfaip3 at eight months. Tnfaip3 was selected for phenotypic testing due to differential expression and the presence of two nonsynonymous mutations. However, when ONL thickness was compared in eight-month-old congenic Tnfaip3(+/-) and Tnfaip3(+/+) mice, no differences were found, suggesting that Tnfaip3 is not the quantitative trait gene (QTG) for the Chr 10 QTL. The GO analysis revealed that GO terms associated with stress and cell remodeling are overrepresented in the ageRD-sensitive C strain compared with the B6a strain with age (eight months). In the ageRD-resistant B6a strain, compared with the C strain, GO terms associated with antioxidant response and the regulation of blood vessel size are overrepresented with age.The analyses of differentially expressed genes and the PosMed database yielded candidate genes for the Chr 6 and Chr 10 QTL. HtrA serine peptidase 2 (Htra2), Dok1, and Tnfaip3 were deemed most promising because of their known roles in apoptosis and our finding of nonsynonymous substitutions between B6a and C strains. While Tnfaip3 was excluded as the QTG for the Chr 10 QTL, Dok1 and Htra2 remain good candidates for the Chr 6 QTL. Finally, the GO term analysis further supports the general hypothesis that oxidative stress is involved in ageRD.

    View details for Web of Science ID 000279676700001

    View details for PubMedID 20577653

    View details for PubMedCentralID PMC2890552

  • Rescue of Glaucoma-Causing Mutant Myocilin Thermal Stability by Chemical Chaperones ACS CHEMICAL BIOLOGY Burns, J. N., Orwig, S. D., Harris, J. L., Watkins, J. D., Vollrath, D., Lieberman, R. L. 2010; 5 (5): 477-487


    Mutations in myocilin cause an inherited form of open angle glaucoma, a prevalent neurodegenerative disorder associated with increased intraocular pressure. Myocilin forms part of the trabecular meshwork extracellular matrix presumed to regulate intraocular pressure. Missense mutations, clustered in the olfactomedin (OLF) domain of myocilin, render the protein prone to aggregation in the endoplasmic reticulum of trabecular meshwork cells, causing cell dysfunction and death. Cellular studies have demonstrated temperature-sensitive secretion of myocilin mutants, but difficulties in expression and purification have precluded biophysical characterization of wild-type (wt) myocilin and disease-causing mutants in vitro. We have overcome these limitations by purifying wt and select glaucoma-causing mutant (D380A, I477N, I477S, K423E) forms of the OLF domain (228-504) fused to a maltose binding protein (MBP) from E. coli . Monomeric fusion proteins can be isolated in solution. To determine the relative stability of wt and mutant OLF domains, we developed a fluorescence thermal stability assay without removal of MBP and provide the first direct evidence that mutated OLF is folded but less thermally stable than wt. We tested the ability of seven chemical chaperones to stabilize mutant myocilin. Only sarcosine and trimethylamine N-oxide were capable of shifting the melting temperature of all mutants tested to near that of wt OLF. Our work lays the foundation for the identification of tailored small molecules capable of stabilizing mutant myocilin and promoting secretion to the extracellular matrix, to better control intraocular pressure and to ultimately delay the onset of myocilin glaucoma.

    View details for DOI 10.1021/cb900282e

    View details for Web of Science ID 000277865500006

    View details for PubMedID 20334347

    View details for PubMedCentralID PMC2874607

  • Mertk Drives Myosin II Redistribution during Retinal Pigment Epithelial Phagocytosis INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE Strick, D. J., Feng, W., Vollrath, D. 2009; 50 (5): 2427-2435


    Mertk is a key phagocytic receptor in the immune, male reproductive, and visual systems. In the retinal pigment epithelium, Mertk is required for the daily ingestion of photoreceptor outer segment (OS) tips. Loss of Mertk function causes retinal degeneration in rats, mice, and humans; however, little is known about the mechanism by which Mertk regulates the ingestion phase of retinal pigment epithelial (RPE) phagocytosis. To address this, the authors sought proteins that associated with Mertk during OS phagocytosis.Lysates of RPE-J cells challenged with OS for various times were immunoprecipitated with Mertk antibody. Potential interacting proteins were identified by mass spectrometry and characterized with confocal microscopy, pharmacologic inhibition, and siRNA knockdown coupled with an in vitro phagocytic assay in primary RPE cells.Myh9, the non-muscle myosin II-A heavy chain, was enriched in immunoprecipitates from OS-treated samples. Myosin II-A and II-B isoforms exhibited a striking redistribution in wild-type rat primary RPE cells challenged with OS, moving from the cell periphery to colocalize with ingested OS over time. In contrast, myosin II-A redistribution in response to OS was blunted in primary RPE cells from RCS rats, which lack functional Mertk. Wild-type rat primary RPE cells treated with the myosin II-specific inhibitor blebbistatin or myosin II siRNAs exhibited a significant phagocytic defect.Mertk mobilizes myosin II from the RPE cell periphery to sites of OS engulfment, where myosin II function is essential for the normal phagocytic ingestion of OS.

    View details for DOI 10.1167/iovs.08-3058

    View details for PubMedID 19117932

  • Sustained Delivery of NT-3 from Lens Fiber Cells in Transgenic Mice Reveals Specificity of Neuroprotection in Retinal Degenerations JOURNAL OF COMPARATIVE NEUROLOGY LaVail, M. M., Nishikawa, S., Duncan, J. L., Yang, H., Matthes, M. T., Yasumura, D., Vollrath, D., Overbeek, P. A., Ash, J. D., Robinson, M. L. 2008; 511 (6): 724-735


    Several neurotrophic factors (NTFs) are effective in protecting retinal photoreceptor cells from the damaging effects of constant light and slowing the rate of inherited photoreceptor degenerations. It is currently unclear whether, if continuously available, all NTFs can be protective for many or most retinal degenerations (RDs). We used transgenic mice that continuously overexpress the neurotrophin NT-3 from lens fibers under the control of the alphaA-crystallin promoter to test for neuroprotection in light-damage experiments and in four naturally occurring or transgenically induced RDs in mice. Lens-specific expression of NT-3 mRNA was demonstrated both by in situ hybridization in embryos and by reverse-transcriptase polymerase chain reaction (RT-PCR) in adult mice. Furthermore, NT-3 protein was found in abundance in the lens, ocular fluids, and retina by enzyme-linked immunosorbent assay (ELISA) and immunocytochemistry. Overexpression of NT-3 had no adverse effects on the structure or function of the retina for up to at least 14 months of age. Mice expressing the NT-3 transgene were protected from the damaging effects of constant light to a much greater degree than those receiving bolus injections of NT-3. When the NT-3 transgene was transferred into rd/rd, Rds/+, Q344ter mutant rhodopsin or Mertk knockout mice, overexpression of NT-3 had no protective effect on the RDs in these mice. Thus, specificity of the neuroprotective effect of NT-3 is clearly demonstrated, and different molecular mechanisms are inferred to mediate the protective effect in light-induced and inherited RDs.

    View details for DOI 10.1002/cne.21858

    View details for Web of Science ID 000260842300002

    View details for PubMedID 18925574

    View details for PubMedCentralID PMC2702994

  • Rapid and stable knockdown of an endogenous gene in retinal pigment epithelium HUMAN GENE THERAPY Paskowitz, D. M., Greenberg, K. P., Yasumura, D., Grimm, D., Yang, H., Duncan, J. L., Kay, M. A., LaVail, M. M., Flannery, J. G., Vollrath, D. 2007; 18 (10): 871-880


    The selective silencing of target genes in specific cell types by RNA interference (RNAi) represents a powerful approach both to gene therapy of dominantly active mutant alleles, and to the investigation of normal gene function in animal models in vivo. We established a simple and versatile in vitro method for screening the efficacy of DNA-based short hairpin RNAs (shRNAs), and identified a highly effective shRNA targeting basic fibroblast growth factor (bFGF), a gene thought to play important roles in endogenous neuroprotective responses in the rat retina. We used two viral vectors, based on lentivirus and adeno-associated virus (AAV), to deliver shRNAs and silence bFGF in retinal pigment epithelial cells in vivo. The AAV experiments made use of a "stabilized double-stranded" version of these vectors with rapid onset of gene expression. In the rat retinal pigment epithelium, shRNAs delivered by either vector reduced bFGF immunoreactivity to undetectable levels in transduced cells, whereas a nonfunctional control construct incorporating a two-base pair mutation had no measurable effect on bFGF expression. Silencing commenced within a few days after injection of virus and remained stable throughout the period of observation, as long as 60 days. Viral delivery of RNAi constructs offers a powerful and versatile approach for both gene therapy and the analysis of fundamental questions in retinal biology.

    View details for DOI 10.1089/hum.2007.065

    View details for Web of Science ID 000250339500001

    View details for PubMedID 17892416

  • Nail-patella syndrome and its association with glaucoma: a review of eight families BRITISH JOURNAL OF OPHTHALMOLOGY Mimiwati, Z., Mackey, D. A., Craig, J. E., MacKinnon, J. R., Rait, J. L., Liebelt, J. E., Ayala-Lugo, R., Vollrath, D., Richards, J. E. 2006; 90 (12): 1505-1511


    Nail-patella syndrome (NPS) is a rare autosomal dominant syndrome, characterised by dysplasia of the nails, patellae, elbows and iliac horns. Mutations in the LMX1B gene were found in four North American families in whom glaucoma cosegregated with NPS.To investigate the association of glaucoma with NPS in Australian families and to determine how common NPS is in Australia.One family with NPS and glaucoma was identified from the Glaucoma Inheritance Study in Tasmania. A further 18 index cases of NPS were identified from the genetics database for southeastern Australia. Eight of these pedigrees were available for comprehensive glaucoma examination on available family members. DNA was sequenced for mutations in LMX1B.In total, 52 living cases of NPS were identified suggesting a minimum prevalence of at least 1 in 100 000. 32 subjects from eight NPS pedigrees (four familial and four sporadic cases) were examined. 14 subjects had NPS alone. 4 subjects had NPS and glaucoma or ocular hypertension. Five pedigrees with NPS had a reported family history of glaucoma, although some of these people with glaucoma did not have NPS. LMX1B mutations were identified in 5 of the 8 index cases-three sporadic and two familial. Two of the six (33%) participants over 40 years of age had developed glaucoma, showing increased risk of glaucoma in NPS.Patients with NPS should be examined regularly for glaucoma. However, because the families with NPS are ascertained primarily from young probands or probands who are isolated cases, the exact level of risk is unclear.

    View details for DOI 10.1136/bjo.2006.092619

    View details for Web of Science ID 000242133800017

    View details for PubMedID 16825280

    View details for PubMedCentralID PMC1857543

  • A novel His158Arg mutation in TIMP3 causes a late-onset form of Sorsby fundus dystrophy AMERICAN JOURNAL OF OPHTHALMOLOGY Lin, R. J., Blumenkranz, M. S., Binkley, J., Wu, K., Vollrath, D. 2006; 142 (5): 839-848


    To describe the phenotype and genotype of a family with suspected Sorsby fundus dystrophy (SFD).Case reports and results of deoxyribonucleic acid (DNA) analysis.Clinical features were determined by complete ophthalmologic examination or by review of medical records. Mutational analysis of the tissue inhibitor of metalloproteinase (TIMP)3 gene was performed by DNA resequencing. Biochemical properties of the mutant TIMP3 protein were studied, and phylogenetic and molecular modeling analyses of TIMP proteins were performed.Fundi of four affected family members demonstrated active or regressed bilateral choroidal neovascularization, whereas another affected individual displayed severe diffuse pigmentary degeneration associated with nyctalopia characteristic of SFD. Onset of disease occurred in the fifth to seventh decades of life. A heterozygous His158Arg mutation was found in seven affected family members and was absent from an unaffected member and 98 unrelated controls. Bioinformatic analyses indicate that histidine 158 is an evolutionarily conserved residue in most vertebrate TIMP homologs and predict that substitution by arginine disrupts TIMP3 function. The mutant protein appears to be expressed by fibroblasts from an affected family member. Molecular modeling suggests that TIMP3 residue 158 may be part of a protein-protein interaction interface.A novel mutation in TIMP3 causes a late-onset form of SFD in this family. His158Arg is the first reported TIMP3 SFD coding sequence mutation that does not create an unpaired cysteine. Further study of this unusual mutation may provide insight into the mechanism of SFD pathogenesis.

    View details for DOI 10.1016/j.ajo.2006.06.003

    View details for Web of Science ID 000242142900019

    View details for PubMedID 16989765

  • Temperature sensitive secretion of mutant myocilins EXPERIMENTAL EYE RESEARCH Vollrath, D., Liu, Y. 2006; 82 (6): 1030-1036


    Recent studies have demonstrated that glaucoma-causing mutant myocilin proteins are misfolded and retained in the endoplasmic reticulum of cells. We showed previously that P370L mutant myocilin is poorly secreted at 37 degrees C and prolonged expression of the protein in differentiated human trabecular meshwork cells results in abnormal morphology and cell killing. Culturing cells at a lower temperature, a condition known to facilitate protein folding, enhances secretion and reverses the cytotoxic effects. We wanted to determine if temperature sensitive secretion is a general property of myocilin missense mutants. Wild-type or mutant forms of myocilin were transiently expressed in HEK 293 cells cultured at either 37 or 30 degrees C and protein secretion was assessed by immunoblotting. Of 15 myocilin missense mutants tested, representing a range in severity of associated glaucoma phenotypes, 14 displayed increased secretion at 30 degrees C. The sole exception was K423E, which is associated with an unusual mode of glaucoma inheritance. Generally, there is an inverse relationship between the degree of mutant myocilin secretion at 30 degrees C and the severity of the associated glaucoma phenotype. Mutants that show abundant secretion at 30 degrees C such as T377M, G364V, I499F and D380A are associated with less virulent glaucoma phenotypes, while mutants such as P370L, I477N, and Y437H display little secretion at 30 degrees C and are associated with more virulent glaucoma phenotypes. We conclude that temperature sensitive secretion is a property of most olfactomedin-domain myocilin mutants. The correlation between temperature sensitive secretion and glaucoma phenotype likely reflects the intrinsic susceptibility to misfolding of individual mutant proteins. These results support the hypothesis that myocilin-induced glaucoma is a protein conformational disease. Facilitating mutant protein folding could be a new approach to development of therapies for this disease.

    View details for DOI 10.1016/j.exer.2005.10.007

    View details for Web of Science ID 000237935200015

    View details for PubMedID 16297911

  • Gene expression profile of human trabecular meshwork cells in response to long-term dexamethasone exposure MOLECULAR VISION Rozsa, F. W., REED, D. M., Scott, K. M., Pawar, H., Moroi, S. E., Kijek, T. G., Krafchak, C. M., Othman, M. I., Vollrath, D., Elner, V. M., Richards, J. E. 2006; 12 (14-15): 125-141


    Topical use of dexamethasone has long been associated with steroid induced-glaucoma, although the mechanism is unknown. We applied a strict filtering of comparative microarray data to more than 18,000 genes to evaluate global gene expression of cultured human trabecular meshwork cells in response to treatment with dexamethasone.Three human trabecular meshwork cell primary cultures from nonglaucomatous donors were incubated with and without dexamethasone for 21 days. Relative gene expression was evaluated by analysis of U133A GeneChip and the results validated using quantitative polymerase chain reaction (PCR).Application of strict filtering to include only genes with statistically significant differences in gene expression across all three trabecular meshwork cell cultures produced a list of 1,260 genes. Significant changes in signal level were observed, including 23 upregulated and 18 downregulated genes that changed greater than three fold in each of three cell cultures. Using quantitative PCR we found changes greater than a thousand fold for two genes (SLP1 and SAA2) and changes greater than a hundred fold for another five genes (ANGPTL7, MYOC, SAA1, SERPINA3, and ZBTB16).Expression changes in trabecular meshwork cells in response to dexamethasone treatment indicate that a group of actins and actin-associated proteins are involved in the development of cross-linked actin networks that form in response to dexamethasone. A trend was identified toward decreased expression of protease genes accompanied by an increased expression of protease inhibitors. Such a trend in nonproteasomal proteolysis conceivably affects gene product levels above the level of transcription. Only two genes, MYOC and IGFBP2, showed significantly elevated expression after dexamethasone treatment in our study and the other three previously published reports of primary culture trabecular meshwork cell gene expression.

    View details for Web of Science ID 000236003200001

    View details for PubMedID 16541013

  • Mutations in TCF8 cause posterior polymorphous corneal dystrophy and ectopic expression of COL4A3 by corneal endothelial cells AMERICAN JOURNAL OF HUMAN GENETICS Krafchak, C. M., Pawar, H., Moroi, S. E., Sugar, A., Lichter, P. R., Mackey, D. A., Mian, S., Nairus, T., Elner, V., Schteingart, M. T., Downs, C. A., Kijek, T. G., Johnson, J. M., Trager, E. H., Rozsa, F. W., Mandal, M. N., Epstein, M. P., Vollrath, D., Ayyagari, R., Boehnke, M., Richards, J. E. 2005; 77 (5): 694-708


    Posterior polymorphous corneal dystrophy (PPCD, also known as PPMD) is a rare disease involving metaplasia and overgrowth of corneal endothelial cells. In patients with PPCD, these cells manifest in an epithelial morphology and gene expression pattern, produce an aberrant basement membrane, and, sometimes, spread over the iris and nearby structures in a way that increases the risk for glaucoma. We previously mapped PPCD to a region (PPCD3) on chromosome 10 containing the gene that encodes the two-handed zinc-finger homeodomain transcription factor TCF8. Here, we report a heterozygous frameshift mutation in TCF8 that segregates with PPCD in the family used to map PPCD3 and four different heterozygous nonsense and frameshift mutations in TCF8 in four other PPCD probands. Family reports of inguinal hernia, hydrocele, and possible bone anomalies in affected individuals suggest that individuals with TCF8 mutations should be examined for nonocular anomalies. We detect transcripts of all three identified PPCD genes (VSX1, COL8A2, and TCF8) in the cornea. We show presence of a complex (core plus secondary) binding site for TCF8 in the promoter of Alport syndrome gene COL4A3, which encodes collagen type IV alpha 3, and we present immunohistochemical evidence of ectopic expression of COL4A3 in corneal endothelium of the proband of the original PPCD3 family. Identification of TCF8 as the PPCD3 gene provides a valuable tool for the study of critical gene regulation events in PPCD pathology and suggests a possible role for TCF8 mutations in altered structure and function of cells lining body cavities other than the anterior chamber of the eye. Thus, this study has identified TCF8 as the gene responsible for approximately half of the cases of PPCD, has implicated TCF8 mutations in developmental abnormalities outside the eye, and has presented the TCF8 regulatory target, COL4A3, as a key, shared molecular component of two different diseases, PPCD and Alport syndrome.

    View details for Web of Science ID 000232569600002

    View details for PubMedID 16252232

    View details for PubMedCentralID PMC1271382

  • phi C31 integrase confers genomic integration and long-term transgene expression in rat retina INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE Chalberg, T. W., Genise, H. L., Vollrath, D., Calos, M. P. 2005; 46 (6): 2140-2146


    Gene therapy has shown promise in animal models of retinal disease, with the most success achieved to date with viral vectors used for gene delivery. Viral vectors, however, have side effects and limitations and are difficult to manufacture. The present study was conducted in an attempt to develop a novel system for long-term gene transfer in rat retinal pigment epithelium (RPE), by using nonviral transfection methods for gene transfer and the integrase from the bacteriophage phiC31 to confer long-term gene expression by means of genomic integration.Efficient nonviral delivery of plasmid DNA to rat RPE in vivo was achieved by using subretinal injection of plasmid DNA, followed by in situ electroporation. Gene delivery was evaluated by analyzing enhanced green fluorescent protein (eGFP) expression in frozen sections. In subsequent experiments, a plasmid expressing luciferase, with or without a plasmid encoding the phiC31 integrase, was delivered to rat RPE. Luciferase expression was followed over time by using in vivo luciferase imaging.Subretinal injection followed by electroporation yielded abundant transgene expression in the rat RPE. Expression was strongest 48 hours after delivery. In the absence of phiC31 integrase, transgene expression declined to near-background levels within 3 to 4 weeks after treatment. By contrast, coinjection of the integrase plasmid led to long-term stable transgene expression throughout the 4.5-month test period. Eyes injected with phiC31 integrase showed approximately 85-fold higher long-term transgene expression in the retina than eyes without integrase.Subretinal injection of DNA followed by electroporation affords abundant transfer of plasmid DNA in rat RPE. phiC31 integrase confers robust long-term transgene expression by mediating genomic integration of the transgene. These findings suggest that phiC31 integrase may be a simple and effective tool for nonviral long-term gene transfer in the eye.

    View details for DOI 10.1167/iovs.04-1252

    View details for Web of Science ID 000229504600038

    View details for PubMedID 15914635

  • Reversal of mutant myocilin non-secretion and cell killing: implications for glaucoma HUMAN MOLECULAR GENETICS Liu, Y. H., Vollrath, D. 2004; 13 (11): 1193-1204


    Glaucoma is a progressive blinding disease characterized by gradual loss of vision due to optic neuropathy and retinal ganglion cell death. Increased intraocular pressure is a common feature of glaucoma that is thought to arise from an increased resistance to outflow of aqueous humor through the trabecular meshwork. Mutations of the myocilin gene are one cause of autosomal dominant juvenile- and adult-onset primary open angle glaucoma, but the mechanism by which mutant myocilins cause disease is poorly understood. We have found that disease-causing myocilin mutants are misfolded, are highly aggregation-prone and accumulate in large aggregates in the endoplasmic reticulum (ER) of human embryonic kidney cells and differentiated primary human trabecular meshwork (HTM) cells. In HTM cells, Pro370Leu mutant myocilin is not secreted under normal culture conditions and prolonged expression results in abnormal cell morphology and cell killing. Culturing HTM cells at 30 degrees C, a condition known to facilitate protein folding, promotes secretion of mutant myocilin, normalizes cell morphology and reverses cell lethality. Our results indicate that myocilin-associated glaucoma is an ER storage disease and suggest a progression of events in which chronic expression of misfolded, non-secreted myocilin leads to HTM cell death, trabecular meshwork dysfunction and, ultimately, a dominant glaucoma phenotype. The beneficial effects of facilitating folding and secretion of mutant myocilin suggest a new type of treatment for this form of glaucoma.

    View details for DOI 10.1093/hmg/ddh128

    View details for Web of Science ID 000221406000010

    View details for PubMedID 15069026

  • MERTK arginine-844-cysteine in a patient with severe rod-cone dystrophy: Loss of mutant protein function in transfected CeRs INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE McHenry, C. L., Liu, Y. H., Feng, W., Nair, A. R., Feathers, K. L., Ding, X. L., Gal, A., Vollrath, D., Sieving, P. A., Thompson, D. A. 2004; 45 (5): 1456-1463


    Mutations in the MERTK gene are responsible for retinal degeneration in the Royal College of Surgeons (RCS) rat and are a cause of human autosomal recessive retinitis pigmentosa (RP). This study reports the identification and functional analysis of novel MERTK mutations to provide information regarding whether they are causative of severe rod-cone degeneration in a young patient.MERTK missense variants identified by single-strand conformational polymorphism (SSCP) and sequence analysis were introduced into expression constructs and used to transfect HEK293T cells. Recombinant protein expression was assayed with anti-MERTK and anti-phosphotyrosine antibodies. Protein turnover was assayed in pulse-chase studies of 35S-methionine incorporation. Transcript levels were determined by quantitative RT-PCR.Three MERTK sequence variants were identified in a patient with rod-cone dystrophy: R722X in exon 16 and R865W in exon 19 on the paternal allele and R844C in exon 19 on the maternal allele. The R844C sequence change affects an evolutionarily conserved amino acid residue and was not detected in unaffected individuals. In transfected HEK293Tcells, wild-type (wt) and W865 MERTK were expressed at equivalent levels and present in the plasma membrane, stimulated tyrosine phosphorylation, and induced significant rounding of the cell bodies. In contrast, C844 MERTK was expressed at low levels and did not stimulate tyrosine phosphorylation. In addition, the relative stability of C844 MERTK was significantly less than wt in assays of protein turnover. At age 13, the patient had 20/60 and 20/200 acuities, tunnel vision of 5 degrees centrally, and a far temporal peripheral crescent bilaterally, and ERGs were nondetectable. The fundi showed bull's-eye macular atrophy and widespread RPE thinning.The present study reports the identification of R844C, the first putative pathogenic MERTK missense mutation that results in severe retinal degeneration with childhood onset when in compound heterozygous form with a R722X allele. The loss of function of C844 MERTK is probably due to decreased protein stability.

    View details for DOI 10.1167/iovs.03-0909

    View details for Web of Science ID 000221084700026

    View details for PubMedID 15111602

  • An RCS-like retinal dystrophy phenotype in Mer knockout mice INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE Duncan, J. L., LaVail, M. M., Yasumura, D., Matthes, M. T., Yang, H. D., Trautmann, N., Chappelow, A. V., Feng, W., Earp, H. S., Matsushima, G. K., Vollrath, D. 2003; 44 (2): 826-838


    To determine whether mice that are homozygous for a targeted disruption of the Mer receptor tyrosine kinase gene (mer(kd)) manifest a retinal dystrophy phenotype similar to RCS rats, which carry a mutation in the orthologous gene MERTK:Eyes of mer(kd) and C57BL/6 wild-type (WT) mice were examined by light and electron microscopy, whole-eye rhodopsin measurement, and Ganzfeld electroretinography (ERG).The mer(kd) mice showed rapid, progressive degeneration of the photoreceptors (PRs). Features of the phenotype common to mer(kd) mice and RCS rats included the absence or near absence of phagosomes in the retinal pigment epithelium (RPE) at the peak of outer segment (OS) disc shedding, accumulation of debris and whorls of membranes at the RPE-OS interface, transient supernormal rhodopsin content and OS lengths, the presence of OS vacuoles beginning at early ages, and a relatively slow removal of pyknotic PR nuclei. Most PRs were missing, and OS debris was removed by approximately postnatal day (P)45. Scotopic ERG responses were lower than age-matched WT responses and declined with PR loss. Photopic responses were preserved better than scotopic responses, corresponding with preferential cone preservation as judged histologically. ERG amplitudes were usually unmeasurable beyond P40, although a small-amplitude scotopic threshold response (STR) could still be elicited at P253 in some mice when only scattered PR nuclei remained.Ablation of Mer function in mer(kd) mice results in a retinal phenotype almost identical with that of RCS rats. The similarity in phenotypes between the two rodent models suggests that an RPE phagocytic defect is a feature of all types of retinal degeneration caused by loss of function of Mer tyrosine kinase, perhaps including mutations in human MERTK.

    View details for DOI 10.1167/iovs.02-0438

    View details for Web of Science ID 000180966800052

    View details for PubMedID 12556419

  • Mertk triggers uptake of photoreceptor outer segments during phagocytosis by cultured retinal pigment epithelial cells JOURNAL OF BIOLOGICAL CHEMISTRY Feng, W., Yasumura, D., Matthes, M. T., LaVail, M. M., Vollrath, D. 2002; 277 (19): 17016-17022


    The RCS rat is a widely studied model of recessively inherited retinal degeneration. The genetic defect, known as rdy (retinal dystrophy), results in failure of the retinal pigment epithelium (RPE) to phagocytize shed photoreceptor outer segment membranes. We previously used positional cloning and in vivo genetic complementation to demonstrate that Mertk is the gene for rdy. We have now used a rat primary RPE cell culture system to demonstrate that the RPE is the site of action of Mertk and to obtain functional evidence for a key role of Mertk in RPE phagocytosis. We found that Mertk protein is absent from RCS, but not wild-type, tissues and cultured RPE cells. Delivery of rat Mertk to cultured RCS RPE cells by means of a recombinant adenovirus restored the cells to complete phagocytic competency. Infected RCS RPE cells ingested exogenous outer segments to the same extent as wild-type RPE cells, but outer segment binding was unaffected. Mertk protein progressively co-localized with outer segment material during phagocytosis by primary RPE cells, and activated Mertk accumulated during the early stages of phagocytosis by RPE-J cells. We conclude that Mertk likely functions directly in the RPE phagocytic process as a signaling molecule triggering outer segment ingestion.

    View details for DOI 10.1074/jbc.M107876200

    View details for PubMedID 11861639

  • Retinal dystrophy due to paternal isodisomy for chromosome 1 or chromosome 2, with homoallelism for mutations in RPE65 or MERTK, respectively AMERICAN JOURNAL OF HUMAN GENETICS Thompson, D. A., McHenry, C. L., Li, Y., Richards, J. E., Othman, M. I., Schwinger, E., Vollrath, D., Jacobson, S. G., Gal, A. 2002; 70 (1): 224-229


    Uniparental disomy (UPD) is a rare condition in which a diploid offspring carries a chromosomal pair from a single parent. We now report the first two cases of UPD resulting in retinal degeneration. We identified an apparently homozygous loss-of-function mutation of RPE65 (1p31) in one retinal dystrophy patient and an apparently homozygous loss-of-function mutation of MERTK (2q14.1) in a second retinal dystrophy patient. In both families, the gene defect was present in the patient's heterozygous father but not in the patient's mother. Analysis of haplotypes in each nuclear kindred, by use of DNA polymorphisms distributed along both chromosomal arms, indicated the absence of the maternal allele for all informative markers tested on chromosome 1 in the first patient and on chromosome 2 in the second patient. Our results suggest that retinal degeneration in these individuals is due to apparently complete paternal isodisomy involving reduction to homoallelism for RPE65 or MERTK loss-of-function alleles. Our findings provide evidence for the first time, in the case of chromosome 2, and confirm previous observations, in the case of chromosome 1, that there are no paternally imprinted genes on chromosomes 1 and 2 that have a major effect on phenotype.

    View details for Web of Science ID 000172571900019

    View details for PubMedID 11727200

    View details for PubMedCentralID PMC384890

  • Molecular and clinical evaluation of a patient hemizygous for TIGR/MYOC ARCHIVES OF OPHTHALMOLOGY Wiggs, J. L., Vollrath, D. 2001; 119 (11): 1674-1678


    To determine if a patient with an interstitial deletion of chromosome 1 is hemizygous for the TIGR/MYOC gene and if that patient has glaucoma.A patient with an interstitial deletion of chromosome 1 was clinically examined for evidence of glaucoma. DNA samples from the patient and her family were used for molecular studies to determine the boundaries of the chromosome 1 deletion using polymorphic markers located on chromosome 1q21 to 1q24. Additional markers located in the vicinity of the TIGR/MYOC gene, including 2 derived from the ends of the gene, were used to determine if it was included in the deletion.The patient and her family showed no evidence of glaucoma. Molecular analysis demonstrated that a complex deletion of the maternal copy of chromosome 1 included the entire TIGR/MYOC gene.We have determined that the patient has only 1 functional copy of TIGR/MYOC. The lack of clinical evidence of glaucoma suggests that haploinsufficiency of the TIGR/MYOC protein is not the cause of early-onset glaucoma associated with mutations in TIGR/MYOC.Missense and nonsense mutations in the TIGR/MYOC gene have been associated with juvenile- and adult-onset primary open-angle glaucoma. Although many different mutations have been correlated with the disease, the underlying genetic mechanism (haploinsufficiency, gain of function, or a dominant negative effect) remains unknown. Information regarding the genetic mechanism responsible for TIGR/MYOC-associated glaucoma is necessary for further studies designed to develop transgenic animal models and gene-related therapy.

    View details for Web of Science ID 000172055600011

    View details for PubMedID 11709019

  • Correction of the retinal dystrophy phenotype of the RCS rat by viral gene transfer of Mertk PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Vollrath, D., Feng, W., Duncan, J. L., Yasumura, D., D'Cruz, P. M., Chappelow, A., Matthes, M. T., Kay, M. A., LaVail, M. M. 2001; 98 (22): 12584-12589


    The Royal College of Surgeons (RCS) rat is a widely studied animal model of retinal degeneration in which the inability of the retinal pigment epithelium (RPE) to phagocytize shed photoreceptor outer segments leads to a progressive loss of rod and cone photoreceptors. We recently used positional cloning to demonstrate that the gene Mertk likely corresponds to the retinal dystrophy (rdy) locus of the RCS rat. In the present study, we sought to determine whether gene transfer of Mertk to a RCS rat retina would result in correction of the RPE phagocytosis defect and preservation of photoreceptors. We used subretinal injection of a recombinant replication-deficient adenovirus encoding rat Mertk to deliver the gene to the eyes of young RCS rats. Electrophysiological assessment of animals 30 days after injection revealed an increased sensitivity of treated eyes to low-intensity light. Histologic and ultrastructural assessment demonstrated substantial sparing of photoreceptors, preservation of outer segment structure, and correction of the RPE phagocytosis defect in areas surrounding the injection site. Our results provide definitive evidence that mutation of Mertk underlies the RCS retinal dystrophy phenotype, and that the phenotype can be corrected by treatment of juvenile animals. To our knowledge, this is the first demonstration of complementation of both a functional cellular defect (phagocytosis) and a photoreceptor degeneration by gene transfer to the RPE. These results, together with the recent discovery of MERTK mutations in individuals with retinitis pigmentosa, emphasize the importance of the RCS rat as a model for gene therapy of diseases that arise from RPE dysfunction.

    View details for Web of Science ID 000171806100054

    View details for PubMedID 11592982

    View details for PubMedCentralID PMC60097

  • Mutations in MERTK, the human orthologue of the RCS rat retinal dystrophy gene, cause retinitis pigmentosa NATURE GENETICS Gal, A., Li, Y., Thompson, D. A., Weir, J., Orth, U., Jacobson, S. G., Apfelstedt-Sylla, E., Vollrath, D. 2000; 26 (3): 270-271


    Mutation of a receptor tyrosine kinase gene, Mertk, in the Royal College of Surgeons (RCS) rat results in defective phagocytosis of photoreceptor outer segments by the retinal pigment epithelium (RPE) and retinal degeneration. We screened the human orthologue, MERTK, located at 2q14.1 (ref. 10), in 328 DNA samples from individuals with various retinal dystrophies and found three mutations in three individuals with retinitis pigmentosa (RP). Our findings are the first conclusive evidence implicating the RPE phagocytosis pathway in human retinal disease.

    View details for Web of Science ID 000165176500010

    View details for PubMedID 11062461

  • Age-dependent prevalence of mutations at the GLC1A locus in primary open-angle glaucoma AMERICAN JOURNAL OF OPHTHALMOLOGY Shimizu, S., Lichter, P. R., Johnson, A. T., Zhou, Z. H., Higashi, M., Gottfredsdottir, M., Othman, M., Moroi, S. E., Rozsa, F. W., Schertzer, R. M., Clarke, M. S., SCHWARTZ, A. L., Downs, C. A., Vollrath, D., Richards, J. E. 2000; 130 (2): 165-177


    To screen a population with primary open-angle glaucoma for mutations in the gene that encodes the trabecular meshwork inducible glucocorticoid response protein (TIGR), also known as myocilin (MYOC).Ophthalmologic information was collected for study subjects with primary open-angle glaucoma and their relatives. Mutation screening of 74 primary open-angle glaucoma probands was conducted by sequencing TIGR/MYOC coding sequence and splice sites.In 23 families we detected 13 nonsynonymous sequence changes, nine of which appear to be mutations likely to cause or contribute to primary open-angle glaucoma. Two mutations, Arg272Gly and Ile499Ser, and one nonsynonymous sequence variant, Asn57Asp, are novel. We found mutations in nine of 25 juvenile glaucoma probands (36%) and two of 49 adult-onset glaucoma probands (4%). Age classification of families rather than individual probands revealed mutations in three of nine families with strictly juvenile primary open-angle glaucoma (33%), and no mutations in 39 families with strictly adult-onset primary open-angle glaucoma (0%). In families with mixed-onset primary open-angle glaucoma containing both juvenile primary open-angle glaucoma and adult-onset primary open-angle glaucoma cases, we found mutations in eight of 26 families (31%).Our data suggest that Gly252Arg, Arg272Gly, Glu323Lys, Gln368STOP, Pro370Leu, Thr377Met, Val426Phe, Ile477Asn, and Ile499Ser are likely to play roles that cause or contribute to the etiology of autosomal dominant primary open-angle glaucoma. Our finding of more TIGR/MYOC mutations in families with mixed-onset primary open-angle glaucoma than in the families with strictly adult-onset primary open-angle glaucoma implies that the presence of relatives with juvenile primary open-angle glaucoma in a family could be used as a basis for identifying a subset of the population with adult-onset primary open-angle glaucoma with higher prevalence of TIGR/MYOC mutations. To address this issue, and to refine estimations of mutation prevalence in these age-defined subpopulations, prospective study of a larger population ascertained entirely through adult-onset primary open-angle glaucoma probands will be needed.

    View details for Web of Science ID 000089601800004

    View details for PubMedID 11004290

  • Mutation of the receptor tyrosine kinase gene Mertk in the retinal dystrophic RCS rat HUMAN MOLECULAR GENETICS D'Cruz, P. M., Yasumura, D., Weir, J., Matthes, M. T., Abderrahim, H., LaVail, M. M., Vollrath, D. 2000; 9 (4): 645-651


    Vertebrate photoreceptor cells are the basic sensory apparatus of the retina, capable of converting the energy of absorbed photons into neuronal signals. The proximal portions of mammalian photoreceptor outer segments are synthesized daily by cell bodies, and outer segment tips are shed with a circadian rhythm, resulting in a complete turnover of outer segments about every 9 days. The shed outer segments are phagocytosed by adjacent retinal pigment epithelial (RPE) cells, and metabolites are recycled to photoreceptors. The Royal College of Surgeons (RCS) rat is a widely studied, classic model of recessively inherited retinal degeneration in which the RPE fails to phagocytose shed outer segments, and photoreceptor cells subsequently die. We have used a positional cloning approach to study the rdy (retinal dystrophy) locus of the RCS rat. Within a 0.3 cM genetic inclusion interval, we have discovered a small deletion of RCS DNA that disrupts the gene encoding the receptor tyrosine kinase Mertk. The deletion includes the splice acceptor site upstream of the second coding exon of Mertk and results in a shortened transcript that lacks this exon. The aberrant transcript joins the first and third coding exons, leading to a frameshift and a translation termination signal 20 codons after the AUG. The concordance of these and other data indicate that Mertk is probably the gene for rdy. Our results provide genetic evidence for an essential role of a receptor tyrosine kinase in a specialized form of phagocytosis and suggest a molecular model for ingestion of outer segments by RPE cells.

    View details for Web of Science ID 000085781000020

    View details for PubMedID 10699188

  • A cellular assay distinguishes normal and mutant TIGR/myocilin protein HUMAN MOLECULAR GENETICS Zhou, Z. H., Vollrath, D. 1999; 8 (12): 2221-2228


    Glaucoma is a blinding eye disease that affects approximately 70 000 000 people world-wide. Mutations in the gene TIGR / MYOC have been shown to cause the most common form of the disease, primary open angle glaucoma, in selected families. Amino acid sequence variants of the gene have been found in 2-4% of sporadic primary open angle glaucoma cases. Most variants are rare and it is often difficult to definitively distinguish between a deleterious mutation and a benign variant solely on the basis of relative frequencies in patient and control groups. The function of the TIGR/myocilin protein is unknown and an assay to functionally classify variants is lacking. We sought to develop a biochemical assay to distinguish different forms of TIGR/myocilin. We investigated the Triton X-100 detergent solubility characteristics of mutant and normal forms of the protein, expressed by transfection in cultured cells. We observed a clear difference in the behavior of the two types of TIGR/myocilin; all confirmed mutant proteins tested were substantially Triton insoluble, while normal protein and controls were completely soluble. We also tested seven ambiguous variant proteins and classified them as mutant or normal on the basis of their Triton solubility. The results in some cases validated, and in other cases contradicted, earlier classifications of these variants. To our knowledge, Triton solubility is the first example of a general difference in the properties of mutant and normal forms of TIGR/myocilin. The assay we have developed will be useful for discerning protein functional information from the location of mutations, will aid genetic counseling of individuals with TIGR/myocilin variants and may provide a clue to understanding a mechanism by which mutations in TIGR / MYOC cause glaucoma.

    View details for Web of Science ID 000083658800011

    View details for PubMedID 10545602

  • Gln368STOP myocilin mutation in families with late-onset primary open-angle glaucoma INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE Allingham, R. R., Wiggs, J. L., De La Paz, M. A., Vollrath, D., Tallett, D. A., Broomer, B., Jones, K. H., del Bono, E. A., Kern, J., Patterson, K., Haines, J. L., Pericak-Vance, M. A. 1998; 39 (12): 2288-2295


    To examine families ascertained for late-onset primary open-angle glaucoma (POAG) to determine mutations in the gene coding for myocilin.The diagnosis of late-onset POAG was defined as age at diagnosis more than 35 years, intraocular pressure (IOP) 22 mm Hg or more in both eyes or 19 mm Hg or more while the patient was taking two glaucoma medications, glaucomatous optic neuropathy in both eyes, and visual field loss consistent with optic nerve damage in at least one eye of the proband. Two of three criteria were required in other family members. DNA from all families was screened for polymorphisms in myocilin using single-strand conformation polymorphism analysis. All polymorphisms were sequenced for mutations.Eighty-three affected people in 29 families with late-onset POAG were screened for mutations. Three mutations, two novel missense (Thr377Met and Glu352Lys) and one nonsense (Gln368STOP), were identified. The missense mutations did not segregate with the disease phenotype in these families. The nonsense mutation was found in 3 of 29 unrelated families with POAG. All affected family members and 8 of 12 in whom glaucoma was suspected had the Gln368STOP mutation. All people with this mutation had elevated IOP, and 78% had POAG by age 70.Three mutations were identified in the gene coding for myocilin in families with late-onset POAG. Of these, the Gln368STOP mutation was highly associated with the development of glaucoma. All people with this mutation had glaucoma or elevated IOP by age 70. In the United States, the Gln368STOP mutation in myocilin is strongly associated with the development of late-onset POAG. However, factors in addition to the presence of this mutation seem to play a role in the development of ocular hypertension and glaucoma in these families.

    View details for Web of Science ID 000076625100012

    View details for PubMedID 9804137

  • Autosomal dominant nanophthalmos (NNO1) with high hyperopia and angle-closure glaucoma maps to chromosome 11 AMERICAN JOURNAL OF HUMAN GENETICS Othman, M. I., Sullivan, S. A., Skuta, G. L., Cockrell, D. A., Stringham, H. M., Downs, C. A., Fornes, A., Mick, A., Boehnke, M., Vollrath, D., Richards, J. E. 1998; 63 (5): 1411-1418


    Nanophthalmos is an uncommon developmental ocular disorder characterized by a small eye, as indicated by short axial length, high hyperopia (severe farsightedness), high lens/eye volume ratio, and a high incidence of angle-closure glaucoma. We performed clinical and genetic evaluations of members of a large family in which nanophthalmos is transmitted in an autosomal dominant manner. Ocular examinations of 22 affected family members revealed high hyperopia (range +7.25-+13.00 diopters; mean +9.88 diopters) and short axial length (range 17.55-19.28 mm; mean 18.13 mm). Twelve affected family members had angle-closure glaucoma or occludable anterior-chamber angles. Linkage analysis of a genome scan demonstrated highly significant evidence that nanophthalmos in this family is the result of a defect in a previously unidentified locus (NNO1) on chromosome 11. The gene was localized to a 14.7-cM interval between D11S905 and D11S987, with a maximum LOD score of 5. 92 at a recombination fraction of .00 for marker D11S903 and a multipoint maximum LOD score of 6.31 for marker D11S1313. NNO1 is the first human locus associated with nanophthalmos or with an angle-closure glaucoma phenotype, and the identification of the NNO1 locus is the first step toward the cloning of the gene. A cloned copy of the gene will enable examination of the relationship, if any, between nanophthalmos and less severe forms of hyperopia and between nanophthalmos and other conditions in which angle-closure glaucoma is a feature.

    View details for Web of Science ID 000076985200017

    View details for PubMedID 9792868

    View details for PubMedCentralID PMC1377551

  • Characterization of the murine TIGR/myocilin gene MAMMALIAN GENOME Abderrahim, H., Jaramillo-Babb, V. L., Zhou, Z. H., Vollrath, D. 1998; 9 (8): 673-675

    View details for Web of Science ID 000074925000016

    View details for PubMedID 9680392

  • Loss-of-function mutations in the LIM-homeodomain gene, LMX1B, in nail-patella syndrome HUMAN MOLECULAR GENETICS Vollrath, D., Jaramillo-Babb, V. L., Clough, M. V., McIntosh, I., Scott, K. M., Lichter, P. R., Richards, J. E. 1998; 7 (7): 1091-1098


    Nail-patella syndrome (NPS) is an inherited developmental disorder most commonly involving maldevelopment of the fingernails, kneecaps and elbow joints. NPS exhibits wide variation in phenotypic expression within and among families with respect to these features. Other skeletal abnormalities such as hip dislocation and club foot have also been reported in some individuals with NPS. There is an association between NPS and renal disease, and between NPS and open-angle glaucoma (OAG), but it is not known whether mutations in a single gene cause the observed skeletal, renal and ophthalmic abnormalities. Recently, LMX1B , a transcription factor of the LIM-homeodomain type with homologs that are important for limb development in vertebrates, was mapped to the same general location as NPS at 9q34. We sequenced a large segment of LMX1B from the genomic DNA of probands from four families with NPS and OAG, and identified four mutations: two stop codons, a deletion causing a frameshift and a missense mutation in a functionally important residue. The presence of these putative loss-of-function mutations in the DNA of individuals with NPS indicates that haploinsufficiency of LMX1B underlies this disorder. These findings help to explain the high degree of variability in the NPS phenotype, and suggest that the skeletal defects in NPS are a result of the diminished dorsoventral patterning activity of LMX1B protein during limb development. The results further suggest that the NPS and OAG phenotypes in the families studied result from mutations in a single gene, LMX1B.

    View details for Web of Science ID 000074571900004

    View details for PubMedID 9618165

  • Genome maps .7. The human transcript map SCIENCE Schuler, G. D., Boguski, M. S., Hudson, T. J., Hui, L., Ma, J., Castle, A. B., Wu, X., Silva, J., Nusbaum, H. C., Birren, B. B., Slonim, D. K., Rozen, S., Stein, L. D., Page, D., Lander, E. S., Stewart, E. A., Aggarwal, A., Bajorek, E., Brady, S., Chu, A., Fang, N., Hadley, D., Harris, M., Hussain, S., Maratukulam, A., Perkins, S., Piercy, M., Qin, F., Reif, T., Sanders, C., She, X., Sun, W. L., Tabar, P., Voyticky, S., Mader, C., McKusick, K. B., Fan, J. B., Cowles, S., Quackenbush, J., Vollrath, D., Myers, R. M., Cox, D. R., Butler, A., Clee, C., Dibling, T., East, C., Edwards, C., Garrett, C., Green, L., Harrison, P., Hicks, A., Holloway, E., Ranby, S., MacGilvery, A., Mungall, A., Peck, A., Wilmer, T., Soderlund, C., Rice, K., Dunham, I., Bentley, D., Deloukas, P., Gyapay, G., Chiannilkulchai, N., Fizames, C., Bentolila, S., Duprat, S., VegaCzarny, N., Muselet, D., Drouot, N., Morissette, J., Weissenbach, J., Morissette, J., James, M. R., White, R. E., Thangarajah, T., LouisDitSully, C., Day, P. J., Goodfellow, P. N., Schmitt, K., Walter, N. A., Berry, R., IORIO, K. R., Sikela, J. M., Polymeropoulos, M. H., Torres, R., Ide, S. E., Dehejia, A., Houlgatte, R., Auffray, C., Adams, M. D., Phillips, C., Brandon, R., Sandusky, M., Venter, J. C., Seki, N., Nagase, T., Ishikawa, K., Nomura, N., RODRIGUEZTOME, P., Matise, T. C., Lee, W. Y., Swanson, K. A., Hudson, J. R. 1996; 274 (5287): 547-558
  • A gene map of the human genome SCIENCE Schuler, G. D., Boguski, M. S., Stewart, E. A., Stein, L. D., Gyapay, G., Rice, K., White, R. E., RODRIGUEZTOME, P., Aggarwal, A., Bajorek, E., Bentolila, S., Birren, B. B., Butler, A., Castle, A. B., Chiannilkulchai, N., Chu, A., Clee, C., Cowles, S., Day, P. J., Dibling, T., Drouot, N., Dunham, I., Duprat, S., East, C., Edwards, C., Fan, J. B., Fang, N., Fizames, C., Garrett, C., Green, L., Hadley, D., Harris, M., Harrison, P., Brady, S., Hicks, A., Holloway, E., Hui, L., Hussain, S., LouisDitSully, C., Ma, J., MacGilvery, A., Mader, C., Maratukulam, A., Matise, T. C., McKusick, K. B., Morissette, J., Mungall, A., Muselet, D., Nusbaum, H. C., Page, D. C., Peck, A., Perkins, S., Piercy, M., Qin, F., Quackenbush, J., Ranby, S., Reif, T., Rozen, S., Sanders, C., She, X., Silva, J., Slonim, D. K., Soderlund, C., Sun, W. L., Tabar, P., Thangarajah, T., VegaCzamy, N., Vollrath, D., Voyticky, S., Wilmer, T., Wu, X., Adams, M. D., Auffray, C., Walter, N. A., Brandon, R., Dehejia, A., Goodfellow, P. N., Houlgatte, R., Hudson, J. R., Ide, S. E., IORIO, K. R., Lee, W. Y., Seki, N., Nagase, T., Ishikawa, K., Nomura, N., Phillips, C., Polymeropoulos, M. H., Sandusky, M., Schmitt, K., Berry, R., Swanson, K., Torres, R., Venter, J. C., Sikela, J. M., Beckmann, J. S., Weissenbach, J., Myers, R. M., Cox, D. R., James, M. R., Bentley, D., Deloukas, P., Lander, E. S., Hudson, T. J. 1996; 274 (5287): 540-546


    The human genome is thought to harbor 50,000 to 100,000 genes, of which about half have been sampled to date in the form of expressed sequence tags. An international consortium was organized to develop and map gene-based sequence tagged site markers on a set of two radiation hybrid panels and a yeast artificial chromosome library. More than 16,000 human genes have been mapped relative to a framework map that contains about 1000 polymorphic genetic markers. The gene map unifies the existing genetic and physical maps with the nucleotide and protein sequence databases in a fashion that should speed the discovery of genes underlying inherited human disease. The integrated resource is available through a site on the World Wide Web at

    View details for Web of Science ID A1996VN91900037

    View details for PubMedID 8849440



    We report the discovery of a polymorphic A to G transition found on the human Y chromosome by sequencing Y-specific sequence-tagged sites (STSs). It shows maximal linkage disequilibrium with a previously described Alu insertional polymorphism. We analyze further an apparently African Y chromosome which seems to have entered a Mexican Mayan population several generations ago. Using the newly discovered transition and the Y-specific polymorphic Alu insertion, we discuss how the chromosome's haplotype information might be used to answer questions of human origins and migrations.

    View details for Web of Science ID A1994PY48100009

    View details for PubMedID 7881413

  • TANDEM ARRAY OF HUMAN VISUAL PIGMENT GENES AT XQ28 SCIENCE Vollrath, D., Nathans, J., Davis, R. W. 1988; 240 (4859): 1669-1672


    Unequal crossing-over within a head-to-tail tandem array of the homologous red and green visual pigment genes has been proposed to explain the observed variation in green-pigment gene number among individuals and the prevalence of red-green fusion genes among color-blind subjects. This model was tested by probing the structure of the red and green pigment loci with long-range physical mapping techniques. The loci were found to constitute a gene array with an approximately 39-kilobase repeat length. The position of the red pigment gene at the 5' edge of the array explains its lack of variation in copy number. Restriction maps of the array in four individuals who differ in gene number are consistent with a head-to-tail configuration of the genes. These results provide physical evidence in support of the model and help to explain the high incidence of color blindness in the human population.

    View details for Web of Science ID A1988N833700035

    View details for PubMedID 2837827



    Electric fields can be manipulated by a method in which multiple electrodes are arranged along a closed contour and clamped to predetermined electric potentials. This method may be applied to a broad range of problems in the separation of macromolecules by gel electrophoresis. DNA molecules as large as 2 megabases can be well separated with a contour-clamped homogeneous electric field alternating between two orientations 120 degrees apart. The pattern of separation is independent of position in the gel, which is an advantage over previous methods. DNA less than 50 kilobases can be separated without distortion even at high voltage with a nonalternating contour-clamped homogeneous field. Decreased band broadening in DNA less than 200 bases can be achieved with a contour-clamped inhomogeneous field.

    View details for Web of Science ID A1986F186700038

    View details for PubMedID 3538420