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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-
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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
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