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Letters IOVS, December 2010, Vol. 51, No. 12 and clinical characterizations. Invest Ophthalmol Vis. Sci. 2010; 51(11):5943–5951. 2. Koenekoop RK, Lopez I, den Hollander AI, et al. Genetic testing for retinal dystrophies and dysfunctions: benefits, dilemmas and solutions. Clin Exp Ophthalmol. 2007;35(5):473– 485. 3. Stone EM. Genetic testing for inherited eye disease. Arch Ophthalmol. 2007;125(2):205–212. 6905 Karin W. Littink1,2 Robert K. Koenekoop3 L. Ingeborgh van den Born1 Frans P. M. Cremers2,4 Anneke I. den Hollander2,5 1 The Rotterdam Eye Hospital, Rotterdam, The Netherlands; the Department of Human Genetics, the 4Nijmegen Centre for Molecular Life Sciences, and the 5Department of Ophthalmology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands; and the 3McGill Ocular Genetics Laboratory, Montreal Children’s Hospital Research Institute, McGill University Health Centre, Montreal, Canada. E-mail: [email protected] 2 Citation: Invest Ophthalmol Vis Sci. 2010;51:6904 – 6905. doi:10.1167/iovs.10-6145 Author Response: Genetic Testing and Clinical Characterization of Patients with Cone–Rod Dystrophy 1 We appreciate the critical reading of our article by Drs. Thiadens and Klaver, and we fully agree with their statements that good collaboration among clinicians, molecular geneticists, and epidemiologists is warranted, to establish meaningful genotype–phenotype correlations in patients with retinal dystrophies. Drs. Thiadens and Klaver question the gene frequencies provided in our study and point out that the genetic testing was not identical for the entire study group. We would like to comment that our study1 was not an epidemiologic study and has not been presented as such. As stated in the study’s introduction, the goal was to identify and map homozygous regions in a large cohort of patients with cone–rod dystrophy (CRD). We clearly explained in the Methods and in the Results sections that only part of the patients have been screened for known mutations in ABCA4. Moreover, it is obvious that homozygosity mapping can detect only homozygous mutations. Homozygous mutations are detected in ⬃35% of Western European patients with autosomal recessive diseases and consequently we did not expect that homozygosity mapping would allow us to detect mutations in the entire study group, as also mentioned in the introduction of the article. Perhaps we should not have presented the percentages of CRD cases that could be attributed to the CRD genes, since not all patients were screened for the complete genes. On the other hand, genetic testing of entire genes in large patient cohorts has always been expensive and laborious, and only limited articles present an accurate overview of percentages of mutations in all genes associated with a specific retinal dystrophy2 or in one specific gene.3 Our study does indicate, however, that mutations in PROM1, CERKL, and EYS are likely to be infrequent causes of CRD. Furthermore, Drs. Thiadens and Klaver mention that the diagnosis of the patients in this cohort may be uncertain. Such uncertainty may be true to some extent, as sometimes the final diagnosis changes over the years with progression of the disease or with the development of systemic features. In a follow-up study of 75 patients with an initial diagnosis of Leber congenital amaurosis, the diagnosis was revised at a later stage to a different disease in 30 patients.4 This actually emphasizes the value of molecular genetic testing to aid a correct diagnosis. Genetic technologies are developing at a tremendous pace, and it is currently possible to screen all retinal dystrophy genes in a patient’s DNA in one experiment, by using a resequencing chip5 or next-generation sequencing (NGS). We are arriving in an era in which it may even be faster to perform genetic screening of all retinal dystrophy genes in a patient’s DNA, rather than to await the results of clinical tests that, particularly in young children, can be challenging or impossible to perform. Besides aiding the diagnosis, we expect that NGS will bring accurate epidemiologic data of mutation frequencies in the near future. References 1. Littink KW, Koenekoop RK, van den Born LI, et al. Homozygosity mapping in patients with cone–rod dystrophy: novel mutations and reappraisal of the clinical diagnosis. Invest Ophthalmol Vis Sci. 2010;51(11):5943–5951. 2. Sullivan LS, Bowne SJ, Birch DG, et al. Prevalence of diseasecausing mutations in families with autosomal dominant retinitis pigmentosa: a screen of known genes in 200 families. Invest Ophthalmol Vis Sci. 2006;47:3052–3064. 3. Littink KW, van den Born LI, Koenekoop RK, et al. Mutations in the EYS gene account for ⬃5% of autosomal recessive retinitis pigmentosa and cause a fairly homogeneous phenotype. Ophthalmology. 2010;117;2026 –2033. 4. Lambert SR, Kriss A, Taylor D, Coffey R, Pembrey M. Follow-up and diagnostic reappraisal of 75 patients with Leber’s congenital amaurosis. Am J Ophthalmol. 1989;107:624 – 631. 5. Booij JC, Bakker A, Kulumbetova J, et al. Simultaneous mutation detection in 90 retinal disease genes in multiple patients using a custom-designed 300-kb retinal resequencing chip. Published online August 27, 2010. Citation: Invest Ophthalmol Vis Sci. 2010;51:6905. doi:10.1167/iovs.10-6565 Effects of Fibroblastic and Endothelial Extracellular Matrices on Corneal Endothelial Cells In their excellent recent report, published online as a recently accepted paper on July 14, 2010, Gruschwitz et al.1 understandably omitted a 25-year-old paper of ours.2 In it, Hseih and I isolated extracellular matrices (ECMs) from chick embryo fibroblasts and human and rabbit corneal stromal cells. These cells induced polarization and elongation of corneal endothelial cells in culture. By indirect immunofluorescence, fibronectin was seen as arrays of long fibers in fibroblastic ECM, whereas in endothelial ECM, fibronectin was found in discrete foci of short fibers. The morphology of endothelial cells in culture was associated with the structure of the ECM that was laid down: short fibers in clusters associated with a typical polygonal shape, with long polarized fibers inducing a fibroblastic appearance. My coworkers and I applaud the authors and look forward to their future efforts. Jules Baum Department of Ophthalmology, Boston University School of Medicine, Boston, Massachusetts. E-mail: [email protected] References 1. Gruschwitz R, Friedrichs J, Valtink M, et al. Alignment and cellmatrix interactions of human corneal endothelial cells on nano- Downloaded From: http://arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/932969/ on 05/06/2017 6906 Letters structured collagen type 1 matrices. Invest Ophthalmol and Vis Sci. 2010;51:6303– 6310. 2. Hsieh P, Baum J. Effects of fibroblastic and endothelial extracellular matrices on corneal endothelial cells. Invest Ophthalmol Vis Sci. 1985;26:457– 463. Citation: Invest Ophthalmol Vis Sci. 2010;51:6905– 6906. doi:10.1167/iovs.10-6230 Author Response: Effects of Fibroblastic and Endothelial Extracellular Matrices on Corneal Endothelial Cells We thank Dr. Baum for his kind, encouraging words and for pointing out his very interesting article on how matrix influences HCEC morphology.1 His work is of great importance to the field of corneal endothelial research. It had a major impact on our earlier work on establishing and optimizing cell cultivation and transplantation of HCECs—namely, his article, “Mass Culture of Human Corneal Endothelial Cells,”2,3 which we cited in one of our most important papers.3 Rita Gruschwitz1 Jens Friedrichs2,3 Monika Valtink1 Clemens Franz4 Daniel J. Müller2,3,5 Richard H. W. Funk1,5 Katrin Engelmann5,6 1 Institute of Anatomy, 2Cellular Machines Group, Biotechnology Center, and 5CRTD/DFG-Center for Regenerative Therapies Dresden–Cluster of Excellence, TU Dresden, Dresden, Germany; 3Department of Biosystems, Science, and Engineering, the Swiss Federal Institute of Technology (ETH) Zurich, Basel, Switzerland; the 4DFG Center for Functional Nanostructures, Institute of Technology, University of Karlsruhe, Karlsruhe, Germany; and 6Department of Ophthalmology, Klinikum Chemnitz GmbH, Chemnitz, Germany. E-mail: [email protected] IOVS, December 2010, Vol. 51, No. 12 acizumab neutralized the secreted VEGF, rather than suppressed it. 2. It is not clear why, even though bevacizumab may have neutralized the secreted VEGF, the neutralized VEGF could not be measured by ELISA. The epitopes for antibodies used in ELISAs (27-191 amino acids) are likely to be against a different region of VEGF than are the functional epitopes bound by bevacizumab. 3. The authors state that VEGF secretion was slightly suppressed with 10 g/mL bevacizumab (P ⫽ 0.0046) and greatly reduced with 100 g/mL (P ⫽ 0.0091) in B16LS9 cells, compared with the effect of the IgG1 control treatment. However, they also state that the levels of VEGF after 10 and 100 g/mL bevacizumab were 8.51 and 15.99 pg/mL, respectively. Thus, the levels were higher in cultures treated with a higher dose of bevacizumab (100 g/mL). Also, in Figure 1C, it does not appear that the VEGF levels were decreased significantly by bevacizumab. 4. Several earlier studies (e.g., Ferrara et al.2) reported that bevacizumab does not neutralize mouse VEGF, raising the question of how bevacizumab may have reduced the VEGF levels. Rajesh K. Sharma Sankarathi Balaiya Kakarla V. Chalam Department of Ophthalmology, University of Florida College of Medicine, Jacksonville, Florida. E-mail: [email protected] References 1. Yang H, Jager MJ, Grossniklaus HE. Bevacizumab suppression of establishment of micrometastases in experimental ocular melanoma. Invest Ophthalmol Vis. Sci. 2010;51(6):2835–2842. 2. Ferrara N, Hillan KJ, Novotny W. Bevacizumab (Avastin), a humanized anti-VEGF monoclonal antibody for cancer therapy. Biochem Biophys Res Commun. 2005;333:328 –335. Citation: Invest Ophthalmol Vis Sci. 2010;51:6906. doi:10.1167/iovs.10-6275 References 1. Hsieh P, Baum J. Effects of fibroblastic and endothelial extracellular matrices on corneal endothelial cells. Invest Ophthalmol Vis Sci. 1985;26:457– 463. 2. Baum JL, Niedra R, Davis C, Yue BY. Mass culture of human corneal endothelial cells. Arch Ophthalmol. 1979;97:1136 – 1140. 3. Engelmann K, Böhnke M, Friedl P. Isolation and long-term cultivation of human corneal endothelial cells. Invest Ophthalmol and Vis Sci. 1988;29:1656 –1662. Citation: Invest Ophthalmol Vis Sci. 2010;51:6906. doi:10.1167/iovs.10-6526 Bevacizumab Suppression of Establishment of Micrometastases in Experimental Ocular Melanoma We read with great interest the article entitled, “Bevacizumab Suppression of Establishment of Micrometastases in Experimental Ocular Melanoma” by Yang et al.,1 published in the June issue. We congratulate the authors for this informative study. However, we have a few concerns about the study. 1. In Figure 1, the authors demonstrate that bevacizumab reduced VEGF secretion. However, it is more likely that bev- Author Response: Bevacizumab Suppression of Establishment of Micrometastases in Experimental Ocular Melanoma Bevacizumab is a recombinant humanized monoclonal IgG1 antibody that binds to and inhibits the biological activity of human vascular endothelial growth factor (VEGF) in in vitro and in vivo assay systems. It contains human framework regions and the complementarity-determining regions of a murine antibody that binds to VEGF. When comparing the sequence of human VEGF protein with that of mouse VEGF protein, we found that 86% of the protein sequence in mouse VEGF164 is similar to that of human VEGF165.1 We believe that it is possible for bevacizumab to block human and mouse VEGF. A blocking antibody is defined as an antibody that does not have a reaction when combined with an antigen, but prevents other antibodies from combining with that antigen.2,3 We believe that bevacizumab fits this definition and explains why the neutralized VEGF is not detected by ELISA. The human or mouse VEGF immunoassay (Quantikine; R&D Systems, Minneapolis, MN) that we used in our experiment can detect human VEGF165 and VEGF121 or mouse VEGF164 and VEGF120, but does not detect VEGF189 and VEGF206. VEGF121 and VEGF165 are diffusible proteins that are secreted into the medium. VEGF189 and VEGF206 have high affinity for heparin and Downloaded From: http://arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/932969/ on 05/06/2017