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What Are the Biomarkers for Glaucoma?
SURVEY OF OPHTHALMOLOGY
VOLUME 52 SUPPLEMENT 2 NOVEMBER 2007
What Are the Biomarkers for Glaucoma? Olga Golubnitschaja, PhD,1 and Josef Flammer, MD2 1
Department of Radiology, University of Bonn, Germany; and 2Department of Ophthalmology, University Hospital Basel, Basel, Switzerland
Abstract. A biomarker is a parameter that can objectively be measured and evaluated as an indicator of either normal or pathologic processes, or of a response to therapeutic intervention. Biomarkers can change qualitatively (mutation/s) or quantitatively (expression level). The known mutations are involved in a negligible minority of glaucoma patients. Therefore, quantitative approaches comparing expression levels are currently under our consideration in terms of potential diagnostic purposes in glaucoma. The following molecular pathways have been shown to be affected by glaucoma pathology: stress response, apoptosis, DNA-repair, cell adhesion, tissue remodeling, transcription regulation, multi-drug resistance, and energy metabolism. Furthermore, circulating leukocytes of glaucoma patients demonstrate some constant alterations in expression patterns. Our ultimate goal is the application of such information for diagnostic and potentially even for screening purposes. New technologies such as disease proteomics and transcriptomics open new perspectives for the development of rapid molecular diagnostics and follow-up in glaucoma. (Surv Ophthalmol 52:S155--S161, 2007. Ó 2007 Elsevier Inc. All rights reserved.) Key words. disease-specific gene expression patterns disease transcriptomics and proteomics DNA damage/repair/methylation status gene hunting glaucoma neurodegeneration non-invasive early diagnosis
Biomarkers are molecules with biologically important intra- or intercellular function, an expression or activity of which either causes or is specifically altered in response to corresponding pathologic condition. Biomarkers are molecules that assign specific biological processes. Biomarkers can be measured physically (e.g., body temperature during an infection or the thickness of the nerve fiber layer in glaucoma patients) or biochemically (e.g., protein concentration in urine). The latter is known as a molecular marker. Molecules can change qualitatively (e.g., in case of gene mutation/s) or quantitatively (e.g., in case of an altered gene expression). The role of biomarkers in medicine is to specify molecular alterations/reactions/pathways attributable to concrete pathologic condition. For instance, an enhanced level of superoxide dismutase in maternal serum is used in prenatal diagnostics as the biomarker for Down syndrome due to the excessive expression of genes encoded on fully or partially trisomic chromosome 21.14,30 Those kinds of molecular alterations as mutations, particularly,
in BRCA-genes have been implicated in pathology of familial breast cancer, and currently are used as blood biomarkers for early diagnostics of breast cancer, admittedly with a moderate success.35 Although mutation research in glaucoma detected some attractive potential targets, the mutated genes found, such as TIGR, play a very limited role in the pathogenesis of glaucoma and do not explain the usual clinical picture.6,22,24,26,39,46 Generally, biomarkers should not be necessarily linked to any genetic mutation; both mRNA and protein expression levels possess valuable information about cellular response to distinct pathologic conditions, and, therefore, are frequently used for the selection of specific biomarkers. Indeed, the most important criterion for a felicitous biomarker is its disease specificity. Whereas alteration in expression status of only one gene is almost never disease specific, a biomarker set is particularly valuable for the creation of highly precise diagnostic approaches. For example, although neuronal thread protein (NTP) demonstrates enhanced expression levels in
S155 Ó 2007 by Elsevier Inc. All rights reserved.
0039-6257/07/$--see front matter doi:10.1016/j.survophthal.2007.08.011
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glaucoma patients against controls,18 the same observation has been done also for Down syndrome, Alzheimer disease, and some other diseases,7 indicating axonal lesions as the common feature but giving no information about the corresponding etiology. Simultaneous monitoring of TAU-protein expression alterations distinguishes between the pathologies, because, in contrast to the accumulation of TAU-protein characteristic for Alzheimer disease, glaucoma patients demonstrate no increase in the target protein versus controls.41 Therefore, the selection of a set of key molecules, the expression levels of which are specifically affected by glaucoma, is particularly valuable for the creation of highly precise molecular diagnostic approaches. Are there currently any reliable biomarkers for glaucoma? Unfortunately there are no molecules identified until now which can specifically be attributed to glaucoma pathology. Further, the frequency and incidence of different glaucoma forms vary geographically, depending on race, sex, and age.19,24,33 Have any molecular diagnostic approaches been developed, in order to select a highrisk group, to distinguish between glaucoma forms and to estimate a disease progression? A current lack of those approaches should be clearly acknowledged.
addition, it has been demonstrated that gene expression patterns of both trabecular meshwork and Schlemm’s canal are similar to those of circulating leukocytes.43 Moreover, blood serum is the biggest reservoir of signaling molecules in human being; many metabolites are simultaneously secreted from different types of cells in blood that represents a universal way of communication between cells. Although this communication is extremely complex, one of the known natural sensors affected by tremendous number of metabolites presented in blood serum are circulating leukocytes. The spectrum of the molecules affecting expression patterns in circulating leukocytes is very broad and partially unknown; their regulation is extremely complex and not yet completely understood. However, it is known that the resulting regulation of circulating leukocytes is triggered by altered gene expression patterns on the levels of transcription and translation. The resulting shift in a gene expression pattern referred to a corresponding physiologic/pathologic condition can be measured on both levels. Both vascular and immune components may play a role in pathomechanisms of glaucoma.11 Taking this in consideration, blood proteome research in glaucoma is of value in order to better understand the molecular pathomechanisms involved in glaucoma and for the development of novel biomarkers for glaucoma diagnostics.
Relevance of Gene Expression Patterns in Eye and Blood for Glaucoma Pathology The current unsatisfactory situation with early diagnosis and protection against glaucoma has motivated researchers to look for new solutions using smart biotechnological approaches. The major challenging issues being confronted are disease-specific alterations in molecular pathways and tissue specificity of molecular patterns. Although animal models for glaucoma provide us with useful information about affected molecular pathways and potential targets in eye, they are barely applicable for non-invasive diagnostics on human being. Because blood is easily accessible, its molecular analysis, in reasonable amounts (a few milliliters), may prove practical application in population screening for high-risk subjects. How relevant are gene expression patterns in blood serum and white blood cells for eye? Glaucoma patients frequently exhibit abnormal Tcell subsets and increased titres of serum antibodies to retina and optic nerve proteins; these alterations in the cellular subset indicate that the immune system plays an important role in the initiation and/ or progression of glaucomatous optic neuropathy.48 Therefore, serum antibodies to retina and optic nerve proteins might be considered as potential indicators for diagnosis of glaucoma pathology.21 In
DNA Damage and Repair Capacity in Glaucoma Research work focused on the ex vivo comparative investigations of DNA damage in circulating leukocytes isolated from patients with glaucoma demonstrated significantly enhanced DNA damage compared to both healthy vasospastic and nonvasospastic individuals.28 Comparative comet assay analysis revealed patterns of comets typical for glaucoma patients as shown in Fig. 1. These findings suggest comet assay profiling of DNA damage in circulating leukocytes as a potentially powerful tool for noninvasive molecular diagnostics of glaucoma disease. Comet assay analysis as a suitable tool for biomarkers has also been suggested for Alzheimer disease.27 Comet assay analysis reveals enhanced DNA damage in both high- and normal-tension glaucoma.28 Whether the level of DNA-damage correlates with disease severity remains to be clarified. Further studies should also evaluate whether significant increase in DNA damage of leukocytes of glaucoma patients is caused by either disease specific stress factors, such as local ischemic/ reperfusion events, and/or decreased capacity of DNA-repair machinery. There is some evidence for
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STRESS RESPONSE, APOPTOSIS AND DNA REPAIR
An increased oxidative stress has been well documented under glaucoma pathology. Rat glaucoma models have demonstrated numerous oxidatively modified proteins in retinal protein lysates from ocular hypertensive eyes.42 Consequently altered gene expression patterns, particularly, for stress response factors can be expected. Thus, heat shock 27 kDa protein 1 demonstrated stable expression alterations in retina hypertensive eyes of rat.1 Gene hunting technology carried out in circulating leukocytes of glaucoma patients revealed an up-regulation of both the pro-apoptotic factor p53 and proteolytic enzyme 20S proteasome subunit XAPC7, activity of which is usually increased during reperfusion.18,47 In consensus, both an apoptotic inhibitor surviving and DNA-repair Xeroderma pigmentosum gene C have been found to be downregulated in circulating leukocytes of glaucoma patients.18 ADHESION, BLOOD--BRAIN BARRIER BREAKDOWN, AND TISSUE REMODELING
Fig. 1. Comet assay ex vivo analysis of DNA damage in circulating leukocytes. Top: Comet patterns typical for healthy controls: there are no visible tails and the majority of chromosomal DNA is focused in heads of comets (intact DNA). Bottom: Comet patterns typical for glaucoma patients: in contrast to the control patterns in the figure on the left, there are clearly visible tails (damaged DNA) and heads of comets are diffuse.
both eventualities: simultaneous up-regulation of p53 (stress regulated gene) and down-regulation of XPGC (essential member of DNA-repair machinery) has been ex vivo demonstrated in circulating leukocytes of glaucoma patients18 and represent potential molecular blood markers for the disease.
Key Molecular Pathways Affected by Glaucoma Pathology: Development of Potential Disease Specific Biomarkers The majority of gene products, of which the expressions are altered in glaucomatous pathology, can be well-organized functional groups belonging to distinct molecular pathways as described subsequently.
Gene expression profiles characteristic for an activated adherent function have been identified in circulating leukocytes of glaucoma patients: upregulated transcripts of lymphocyte-IgE-receptor (Fc-epsilon-RII/CD23), T-cell-specific tyrosine kinase (ITK), thromboxan-A2-receptor, and alkalinephosphatase have been detected by subtractive hybridization.15 Adherent circulating leukocytes could be an important contributor to blood--brain barrier (BBB) breakdown observed in glaucoma pathology.20 The subtraction of metalloproteinase MMP-9 and MT1-MMP transcripts highly increased in circulating leukocytes of glaucoma patients provides further evidence for this functional link and indicates pathways involved in extensive tissue remodeling observed in glaucoma.16 Significantly increased protein expression rates of both latent and active forms of MMP-9 and MT1-MMP in circulating leukocytes correlated well with the enhanced levels of transcription and with glaucoma.16 Once activated, both hydrolyses necessarily contribute to remodeling or even degeneration of the tissue whereto they are secreted by circulating leukocytes. This up-regulation might be a consequence of repeated mild ischemia/reperfusion postulated in glaucoma patients. However, the question of whether there is a correlation between increased metaloproteinase (MMP) activity and glaucoma severity should be further clarified. Recently performed comparative gene transcription profiling in trabecular meshwork isolated from postmortem glaucomatous eyes revealed a significant
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up-regulation of several cell adhesion molecules, including platelet endothelial cells adhesion molecule-1 and P-selectin.9 An enhanced deposition of the cell aggregation protein cochlin in trabecular meshwork and Schlemm’s canal of glaucomatous eye4 supports the crucial role of cellular adhesion in glaucoma pathology and favors cell adhesion molecules as potential disease biomarkers. TRANSCRIPTION REGULATION
Dramatically altered transcription patterns in glaucoma were reported by several research groups.1,15,16,18 A reason for this extensive shift in transcription regulation was unclear for a very long time. Very recently a significant up-regulation of the basic transcriptional regulator AP-2ß was attributed to glaucoma pathology: AP-2ß was identified in circulating leukocytes using the smart technology of disease proteomics.17 Protein mapping for circulating leukocytes is demonstrated in Fig. 2. AP-2 proteins play a decisive role, particularly, in morphogenesis of eye. While the activation of AP-1 leads to increased stromelysin (metalloproteinase-3) production in trabecular meschwork cell cultures,13,31 the expression and activity of AP-2 control the activity of the gelatinase B (MMP-9).29 MMP-9 was demonstrated to be highly activated in leukocytes of glaucoma patients and plays an important role in glaucoma pathology.16 Therefore, the conserved up-regulation of AP-2ß in leukocytes of glaucoma patients has been proposed to be an important part of molecular mechanisms of glaucoma pathology.
Fig. 2. Ex vivo protein mapping by 2D-PAGE imaging in circulating leukocytes of glaucoma patient. For this image, silver staining was performed. On average about 10,000 proteins are supposed to be simultaneously expressed in human leukocytes. Currently most sensitive silver staining visualizes not more than 300 spots for further qualitative and quantitative analysis of protein expression patterns.
GOLUBNITSCHAJA AND FLAMMER MULTI-DRUG RESISTANCE
An extensive dysregulation of ABC-transporters has been demonstrated in glaucoma pathology.10,18,49 ABC-transporters (ATP-binding cassette transporters) usually translocate a wide variety of structurally unrelated lipophilic compounds being responsible for drug efflux, and therefore, for multidrug resistance. ABC 1 has been shown to be stably up-regulated in circulating leukocytes of glaucoma patients.49 Joyce et al demonstrated a crucial role of ABC 1 in protection against atherosclerosis.23 An activity of ABC 1 has been shown to have a regulating effect on the endothelial function and stimulate nitric oxide bio-activity in arterial walls.5 The upregulation of ABC 1 in circulating leukocytes of glaucoma patients might indicate the involvement of this gene in chronic vascular dysregulation frequently observed in glaucoma pathology, and has been suggested as a potential marker for early diagnostics of glaucoma.49 ENERGY METABOLISM
Gene hunting in circulating leukocytes of glaucoma patients has revealed down-regulated transcripts of Na þ/Kþ-ATPase.15 An identification of the subtracted transcripts is demonstrated in Fig. 3. Further, the abnormal sodium handling has been proposed to be associated with ocular hypertensives and to contribute to a progression of the optic nerve damage in both normal-tension and high-tension glaucomas.32,36 Naþ/Kþ-ATPase is known to be down-regulated in lymphocytes of patients with acute myocardial infarction,34 and may be one of the reasons for ventricular arrhythmias and coronary artery spasms. Decrease of intracellular potassium concentration and increase of intracellular calcium concentration may play a major role in the
Fig. 3. Subtractive hybridization is one of the most powerful technologies of gene hunting strategies. Here the subtracted sequence (subtr.) is aligned demonstrating 100% homology to human Naþ/Kþ-ATPase. The subtracted sequence was found to be significantly downregulated in circulating leukocytes of glaucoma patients compared to controls.
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Possible Types of Quantitative and Qualitative Molecular Alterations in Glaucoma Type of Molecule Chromosomal DNA, mitochondrial DNA mRNA
Proteins
Metabolites (signaling molecules, amino acids, plasma hormones, etc.)
Possible Type of Alteration 1. 2. 3. 4. 1.
(Oxidative) damage* Mutations* Polymorphism* Methylation status of CpG islands** Multiple alterations in expression patterns*
2. Reduced mRNA editing 1. Multiple alterations in expression patterns* 2. Posttranslational modification* 3. Phosphorylation status* 4. Protein misfolding* Altered profiles*
Detection Technology -
Comet assay Disease genomics PCR, Restriction analysis, etc. Methylation-specific PCR Disease transcriptomics: Subtractive hybridization, Expression array, ReverseTranscriptase-PCR, Real-TimePCR, etc. - Reverse-Transcriptase-PCR - Disease Proteomics: 2D-PAGE, MALDI-TOF, Western-blot, etc. - Western-blot, Activity tests (e.g. Zymography for gelatinases) - Activity tests - Activity tests Disease Metabolomics: Comparative blood plasma metabolites profiling, high performance liquid chromatography, Activity tests, etc.
MALDI-TOF5matrix assisted laser desorption/ionization time-of-flight; PCR5polymerase chain reaction. Types of molecular alterations reported for glaucoma pathology. ** Data indicating this kind of alteration collected in our laboratory, which have not been published until now. *
pathomechanism of coronary artery spasms. Inhibition of Naþ/Kþ-ATPase is one of functional consequences of oxidative membrane damage caused by an increased oxidative stress,40 which is triggered, for example, under ischemia/reperfusion. This affects central thermogenic mechanisms, because Naþ/Kþ-ATPase has been shown to play a key role in cellular energy balance and thermogenesis.37,44 A decreased activity of Naþ/Kþ-ATPase triggers vasospasm.2 Being an important co-factor of cellular ATPases, magnesium can reverse delayed vasospasm and reduce the extent of acute ischemic lesions.45 Magnesium therapy demonstrates beneficial effects in vasospastic syndrome and glaucoma.12
Effect of Glaucoma Medication on the Evaluation of Biomarkers Evaluation of glaucoma biomarkers under treatment conditions is a particular issue in glaucoma research. Indeed, can we use the markers proposed for diagnostics of untreated glaucoma also under glaucoma medication? Are there any reliable markers that can predict the outcome of glaucoma treatment? There are some studies clearly demonstrating that glaucoma medication may definitely mask alterations in crucial biomarkers such as those of the nitric oxide pathway.25 Additionally, toxic side effects of anti-glaucoma drugs on conjunctiva have been reported: histological studies of conjunctival
tissues in patients who underwent a long-term glaucoma treatment demonstrated an abnormal infiltration of inflammatory cells. Consequently, an abnormal expression of inflammatory markers under various anti-glaucomatous treatments has been shown.3 Much progress has been achieved in the development of markers, which can potentially predict the outcome of glaucoma surgery. In the research work done by Souchier et al, surgery success was defined as dropped IOP # 15 mm Hg without any IOP-lowering drugs at 6 months, and conjunctival expression levels of trefoil factor family 1 (TFF1) and MUC5AC as well as HLA-DR in leukocytes have been measured.38 An increased expression of all three molecular markers has been proposed to be a potential predicting factor of successful glaucoma surgery. However, the issue of glaucoma medication markers is a multifarious one and requires more research effort.
Perspectives for Application of Most Potent Biotechnological Tools High-accuracy proteome maps of human body fluids open new perspectives for early molecular diagnostics of chronic disorders. Thus, recent identification of 491 proteins in the tear fluid proteome reveals a large number of proteases and protease inhibitors.8 An unbalanced secretion of
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proteases against their inhibitors might play an important role in pathogenesis of glaucoma as demonstrated, particularly, for MMP-9 up-regulated in blood.16 Until now, tear fluid has not been investigated for the presence of specific biomarkers in glaucoma. However, proteome research clearly demonstrated that tear fluid should generally be considered as an important source for biomarkers. Obviously this is a potential source for glaucoma biomarkers as well. The human differential blood proteome holds the promise of a revolution in epidemiologic screenings, disease diagnostics, and therapeutic monitoring provided major challenges in proteomics and related disciplines can be addressed also in oncology. Altogether about 3,000 and 10,000 protein products have been reported to be usually present in human plasma and circulating leukocytes, respectively. Abundant scientific evidence from disease proteomics suggest that disease-specific protein expression patterns in blood are indicative for many—if not most—human disorders, and might be a powerful tool for early molecular diagnostics of glaucomatous damage. A non-invasive molecular diagnostic approach based on disease specific gene expression patterns in circulating leukocytes has been recently suggested for glaucoma (Golubnitschaja and Flammer, International Patent No. IB02/00648). The test foresees a precise expression profiling of selected genes in circulating leukocytes isolated from fresh blood samples. These genes have been proposed to play a role in glaucoma pathology and belong to following pathways: apoptosis, stress response, the DNA-repair, transcription regulation, drug resistance, tissue remodeling, and degeneration. Currently a clinical application of the test is under consideration, and a nanotechnology, which should provide a possibly easy and cheap routine application of the test, is under development. Possible types of molecular alterations that can be considered for further development of glaucoma markers as well as corresponding detection technologies are demonstrated in Table 1.
circulating leukocytes. No specific exclusion criteria were used. No time limitation was set, and the following years are covered: 1992--2007. Although no language preference was intended, only articles in English were found and used.
Method of Literature Search The literature search was performed in PubMed. The following search words were used: glaucoma and molecular diagnosis, neurodegeneration, DNA damage/ repair/methylation status, gene hunting, disease transcriptomics and proteomics; proteomes of human body fluids; gene expression patterns in glaucoma, glaucoma medication; glaucoma and bio-markers, stress response, apoptosis, DNA-repair, cell adhesion, tissue remodeling, multi-drug resistance, energy metabolism, blood proteome,
References 1. Ahmed F, Brown KM, Stephan DA, et al: Microarray analysis of changes in mRNA levels in the rat retina after experimental elevation of intraocular pressure. Invest Ophthalmol Vis Sci 45:1247--58, 2004 2. Bagrov AY, Dmitrieva RI, Fedorova OV, et al: Endogenous marinobufagenin-like immunoreactive substance. A possible endogenous Na, K-ATPase inhibitor with vasoconstrictor activity. Am J Hypertens 9:982--90, 1996 3. Baudouin C, Garcher C, Haouat N, et al: Expression of inflammatory membrane markers by conjunctival cells in chronically treated patients with glaucoma. Ophthalmology 101:454--60, 1994 4. Bhattacharya SK, Rockwood EJ, Smith SD, et al: Proteomics reveal cochlin deposits associated with glaucomatous trabecular meshwork. J Biol Chem 280:6080--4, 2005 5. Bisoendial RJ, Hovingh GK, Levels JH, et al: Restoration of endothelial function by increasing high-density lipoprotein in subjects with isolated low high-density lipoprotein. Circulation 107:2944--8, 2003 6. Bruttini M, Longo I, Frezzotti P, et al: Mutations in the myocilin gene in families with primary open-angle glaucoma and juvenile open-angle glaucoma. Arch Ophthalmol 121: 1034--8, 2003 7. de la Monte SM, Xu YY, Hutchins GM, et al: Developmental patterns of neuronal thread protein gene expression in Down syndrome. J Neurol Sci 135:118--25, 1996 8. de Souza GA, Godoy LM, Mann M: Identification of 491 proteins in the tear fluid proteome reveals a large number of proteases and protease inhibitors. Genome Biol 7:R72, 2006 9. Diskin S, Kumar J, Cao Z, et al: Detection of differentially expressed glycogenes in trabecular meshwork of eyes with primary open-angle glaucoma. Invest Ophthalmol Vis Sci 47: 1491--9, 2006 10. Esser J, Esser P, Mietz H, et al: Multidrug resistanceassociated proteins in glaucoma surgery. Graefes Arch Clin Exp Ophthalmol 238:727--32, 2000 11. Flammer J, Mozaffarieh M: What is the present pathogenetic concept of glaucomatous optic neuropathy? Surv Ophthalmol 52(Suppl):S162--73, 2007 12. Flammer J, Pache M, Resink T: Vasospasm, its role in the pathogenesis of diseases with particular reference to the eye. Prog Retin Eye Res 20:319--49, 2001 13. Fleenor DL, Pang IH, Clark AF: Involvement of AP-1 in interleukin-1alpha-stimulated MMP-3 expression in human trabecular meshwork cells. Invest Ophthalmol Vis Sci 44: 3494--501, 2003 14. Golubnitschaja O, Fountoulakis M: Gene hunting by substractive hybridisation in down syndrome; correlation with proteomics analysis, Mandel S, (ed): Handbook of Neurochemistry and Molecular Neurobiology, vol. 25: Genomics, Proteomics and the Nervous System. London, UK, Kluwer Academic/Plenum Publishers, ed 3. 2007, pp. 346--58 15. Golubnitschaja O, Wunderlich K, Decker C, et al: Molecular imaging of perfusion disturbances in glaucoma. Amino Acids 23:293--9, 2002 16. Golubnitschaja O, Yeghiazaryan K, Liu R, et al: Increased expression of matrix metalloproteinases in mononuclear blood cells of normal-tension glaucoma patients. J Glaucoma 13:66--72, 2004 17. Golubnitschaja O, Yeghiazaryan K, Wunderlich K, et al: Disease proteomics reveals altered basic gene expression
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regulation in leukocytes of glaucoma patients. ProteomicsClinical Applications; (In press), 2007 Golubnitschaja-Labudova O, Liu R, Decker C, et al: Altered gene expression in lymphocytes of patients with normaltension glaucoma. Curr Eye Res 21:867--76, 2000 Gong G, Kosoko-Lasaki O, Haynatzki GR, et al: Genetic dissection of myocilin glaucoma. Hum Mol Genet 13(Spec No 1):R91--R102, 2004 Grieshaber MC, Flammer J: Does the blood-brain-barrier play a role in glaucoma? Surv Ophthalmol 52(Suppl):S115-21, 2007 Grus FH, Joachim SC, Bruns K, et al: Serum autoantibodies to alpha-fodrin are present in glaucoma patients from Germany and the United States. Invest Ophthalmol Vis Sci 47:968--76, 2006 Herndon LW, Challa P, Allingham RR: Glaucoma in Ghana, West Africa: clinical features and the role of mutations in Myocilin. Ophthalmol Clin North Am 16:631--7, 2003 Joyce CW, Amar MJ, Lambert G, et al: The ATP binding cassette transporter A1 (ABCA1) modulates the development of aortic atherosclerosis in C57BL/6 and apoE-knockout mice. Proc Natl Acad Sci USA 99:407--12, 2002 Kanagavalli J, Krishnadas SR, Pandaranayaka E, et al: Evaluation and understanding of myocilin mutations in Indian primary open angle glaucoma patients. Mol Vis 9: 606--14, 2003 Kotikoski H, Moilanen E, Vapaatalo H, et al: Biochemical markers of the L-arginine-nitric oxide pathway in the aqueous humour in glaucoma patients. Acta Ophthalmol Scand 80:191--5, 2002 Melki R, Idhajji A, Driouiche S, et al: Mutational analysis of the Myocilin gene in patients with primary open-angle glaucoma in Morocco. Ophthalmic Genet 24:153--60, 2003 Migliore L, Fontana I, Colognato R, et al: Searching for the role and the most suitable biomarkers of oxidative stress in Alzheimer’s disease and in other neurodegenerative diseases. Neurobiol Aging 26:587--95, 2005 Moenkemann H, Flammer J, Wunderlich K, et al: Increased DNA breaks and up-regulation of both G(1) and G(2) checkpoint genes p21(WAF1/CIP1) and 14-3-3 sigma in circulating leukocytes of glaucoma patients and vasospastic individuals. Amino Acids 28:199--205, 2005 Mohan R, Rinehart WB, Bargagna-Mohan P, et al: Gelatinase B/lacZ transgenic mice, a model for mapping gelatinase B expression during developmental and injury-related tissue remodeling. J Biol Chem 273:25903--14, 1998 Ognibene A, Ciuti R, Tozzi P, et al: Maternal serum superoxide dismutase (SOD): a possible marker for screening Down syndrome affected pregnancies. Prenat Diagn 19:1058--60, 1999 Pang IH, Fleenor DL, Hellberg PE, et al: Aqueous outflowenhancing effect of tert-butylhydroquinone: involvement of AP-1 activation and MMP-3 expression. Invest Ophthalmol Vis Sci 44:3502--10, 2003 Pechere-Bertschi A, Sunaric-Megevand G, Haefliger IO, et al: Renal sodium handling in patients with normal pressure glaucoma. Clin Sci (Lond) 112:337--64, 2007 Reddy AB, Panicker SG, Mandal AK, et al: Identification of R368H as a predominant CYP1B1 allele causing primary congenital glaucoma in Indian patients. Invest Ophthalmol Vis Sci 44:4200--3, 2003 Salomon P, Mysiak A, Halawa B: [Sodium efflux through lymphocytic cell membranes in patients with acute myocardial infarction]. Pol Arch Med Wewn 100:543--50, 1998
S161 35. Schmutzler RK, Rhiem K, Breuer P, et al: Outcome of a structured surveillance programme in women with a familial predisposition for breast cancer. Eur J Cancer Prev 15:483--9, 2006 36. Schwartz B, Abrahamson R, Takamoto T, et al: Association of ocular pressure and optic disc cup volume with red blood cell sodium-potassium ATPase inhibition. Curr Eye Res 21: 897--905, 2000 37. Silva JE: Thyroid hormone control of thermogenesis and energy balance. Thyroid 5:481--92, 1995 38. Souchier M, Buron N, Lafontaine PO, et al: Trefoil factor family 1, MUC5AC and human leucocyte antigen-DR expression by conjunctival cells in patients with glaucoma treated with chronic drugs: could these markers predict the success of glaucoma surgery? Br J Ophthalmol 90:1366--9, 2006 39. Sripriya S, Uthra S, Sangeetha R, et al: Low frequency of myocilin mutations in Indian primary open-angle glaucoma patients. Clin Genet 65:333--7, 2004 40. Stark G: Functional consequences of oxidative membrane damage. J Membr Biol 205:1--16, 2005 41. Tatton W, Chen D, Chalmers-Redman R, et al: Hypothesis for a common basis for neuroprotection in glaucoma and Alzheimer’s disease: anti-apoptosis by alpha-2-adrenergic receptor activation. Surv Ophthalmol 48(Suppl 1):S25--37, 2003 42. Tezel G, Yang X, Cai J: Proteomic identification of oxidatively modified retinal proteins in a chronic pressureinduced rat model of glaucoma. Invest Ophthalmol Vis Sci 46:3177--87, 2005 43. Tomarev SI, Wistow G, Raymond V, et al: Gene expression profile of the human trabecular meshwork: NEIBank sequence tag analysis. Invest Ophthalmol Vis Sci 44:2588--96, 2003 44. Valdemarsson S, Ikomi-Kumm J, Monti M: Increased lymphocyte thermogenesis in hyperthyroid patients. Role of Na/K pump function. Evaluation of aerobic/anaerobic metabolism. Acta Endocrinol (Copenh) 126:291--5, 1992 45. van den Bergh WM, Dijkhuizen RM, Rinkel GJ: Potentials of magnesium treatment in subarachnoid haemorrhage. Magnes Res 17:301--13, 2004 46. Wiggs JL, Auguste J, Allingham RR, et al: Lack of association of mutations in optineurin with disease in patients with adult-onset primary open-angle glaucoma. Arch Ophthalmol 121. 118--3, 2003 47. Wunderlich K, Golubnitschaja O, Pache M, et al: Increased plasma levels of 20S proteasome alpha-subunit in glaucoma patients: an observational pilot study. Mol Vis 8:431-5, 2002 48. Yang J, Patil RV, Yu H, et al: T cell subsets and sIL-2R/IL-2 levels in patients with glaucoma. Am J Ophthalmol 131:421-6, 2001 49. Yeghiazaryan K, Flammer J, Wunderlich K, et al: An enhanced expression of ABC 1 transporter in circulating leukocytes as a potential molecular marker for the diagnostics of glaucoma. Amino Acids 28:207--11, 2005 The authors reported no proprietary or commercial interest in any product mentioned or concept discussed in this article. Reprint address: O. Golubnitschaja, PhD, Professor and Head of the Division of Molecular/Experimental Radiology, Radiological Hospital, University of Bonn, Sigmund-Freud-Str. 25, D-53105 Bonn, Germany.
VOLUME 52 SUPPLEMENT 2 NOVEMBER 2007
What Are the Biomarkers for Glaucoma? Olga Golubnitschaja, PhD,1 and Josef Flammer, MD2 1
Department of Radiology, University of Bonn, Germany; and 2Department of Ophthalmology, University Hospital Basel, Basel, Switzerland
Abstract. A biomarker is a parameter that can objectively be measured and evaluated as an indicator of either normal or pathologic processes, or of a response to therapeutic intervention. Biomarkers can change qualitatively (mutation/s) or quantitatively (expression level). The known mutations are involved in a negligible minority of glaucoma patients. Therefore, quantitative approaches comparing expression levels are currently under our consideration in terms of potential diagnostic purposes in glaucoma. The following molecular pathways have been shown to be affected by glaucoma pathology: stress response, apoptosis, DNA-repair, cell adhesion, tissue remodeling, transcription regulation, multi-drug resistance, and energy metabolism. Furthermore, circulating leukocytes of glaucoma patients demonstrate some constant alterations in expression patterns. Our ultimate goal is the application of such information for diagnostic and potentially even for screening purposes. New technologies such as disease proteomics and transcriptomics open new perspectives for the development of rapid molecular diagnostics and follow-up in glaucoma. (Surv Ophthalmol 52:S155--S161, 2007. Ó 2007 Elsevier Inc. All rights reserved.) Key words. disease-specific gene expression patterns disease transcriptomics and proteomics DNA damage/repair/methylation status gene hunting glaucoma neurodegeneration non-invasive early diagnosis
Biomarkers are molecules with biologically important intra- or intercellular function, an expression or activity of which either causes or is specifically altered in response to corresponding pathologic condition. Biomarkers are molecules that assign specific biological processes. Biomarkers can be measured physically (e.g., body temperature during an infection or the thickness of the nerve fiber layer in glaucoma patients) or biochemically (e.g., protein concentration in urine). The latter is known as a molecular marker. Molecules can change qualitatively (e.g., in case of gene mutation/s) or quantitatively (e.g., in case of an altered gene expression). The role of biomarkers in medicine is to specify molecular alterations/reactions/pathways attributable to concrete pathologic condition. For instance, an enhanced level of superoxide dismutase in maternal serum is used in prenatal diagnostics as the biomarker for Down syndrome due to the excessive expression of genes encoded on fully or partially trisomic chromosome 21.14,30 Those kinds of molecular alterations as mutations, particularly,
in BRCA-genes have been implicated in pathology of familial breast cancer, and currently are used as blood biomarkers for early diagnostics of breast cancer, admittedly with a moderate success.35 Although mutation research in glaucoma detected some attractive potential targets, the mutated genes found, such as TIGR, play a very limited role in the pathogenesis of glaucoma and do not explain the usual clinical picture.6,22,24,26,39,46 Generally, biomarkers should not be necessarily linked to any genetic mutation; both mRNA and protein expression levels possess valuable information about cellular response to distinct pathologic conditions, and, therefore, are frequently used for the selection of specific biomarkers. Indeed, the most important criterion for a felicitous biomarker is its disease specificity. Whereas alteration in expression status of only one gene is almost never disease specific, a biomarker set is particularly valuable for the creation of highly precise diagnostic approaches. For example, although neuronal thread protein (NTP) demonstrates enhanced expression levels in
S155 Ó 2007 by Elsevier Inc. All rights reserved.
0039-6257/07/$--see front matter doi:10.1016/j.survophthal.2007.08.011
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glaucoma patients against controls,18 the same observation has been done also for Down syndrome, Alzheimer disease, and some other diseases,7 indicating axonal lesions as the common feature but giving no information about the corresponding etiology. Simultaneous monitoring of TAU-protein expression alterations distinguishes between the pathologies, because, in contrast to the accumulation of TAU-protein characteristic for Alzheimer disease, glaucoma patients demonstrate no increase in the target protein versus controls.41 Therefore, the selection of a set of key molecules, the expression levels of which are specifically affected by glaucoma, is particularly valuable for the creation of highly precise molecular diagnostic approaches. Are there currently any reliable biomarkers for glaucoma? Unfortunately there are no molecules identified until now which can specifically be attributed to glaucoma pathology. Further, the frequency and incidence of different glaucoma forms vary geographically, depending on race, sex, and age.19,24,33 Have any molecular diagnostic approaches been developed, in order to select a highrisk group, to distinguish between glaucoma forms and to estimate a disease progression? A current lack of those approaches should be clearly acknowledged.
addition, it has been demonstrated that gene expression patterns of both trabecular meshwork and Schlemm’s canal are similar to those of circulating leukocytes.43 Moreover, blood serum is the biggest reservoir of signaling molecules in human being; many metabolites are simultaneously secreted from different types of cells in blood that represents a universal way of communication between cells. Although this communication is extremely complex, one of the known natural sensors affected by tremendous number of metabolites presented in blood serum are circulating leukocytes. The spectrum of the molecules affecting expression patterns in circulating leukocytes is very broad and partially unknown; their regulation is extremely complex and not yet completely understood. However, it is known that the resulting regulation of circulating leukocytes is triggered by altered gene expression patterns on the levels of transcription and translation. The resulting shift in a gene expression pattern referred to a corresponding physiologic/pathologic condition can be measured on both levels. Both vascular and immune components may play a role in pathomechanisms of glaucoma.11 Taking this in consideration, blood proteome research in glaucoma is of value in order to better understand the molecular pathomechanisms involved in glaucoma and for the development of novel biomarkers for glaucoma diagnostics.
Relevance of Gene Expression Patterns in Eye and Blood for Glaucoma Pathology The current unsatisfactory situation with early diagnosis and protection against glaucoma has motivated researchers to look for new solutions using smart biotechnological approaches. The major challenging issues being confronted are disease-specific alterations in molecular pathways and tissue specificity of molecular patterns. Although animal models for glaucoma provide us with useful information about affected molecular pathways and potential targets in eye, they are barely applicable for non-invasive diagnostics on human being. Because blood is easily accessible, its molecular analysis, in reasonable amounts (a few milliliters), may prove practical application in population screening for high-risk subjects. How relevant are gene expression patterns in blood serum and white blood cells for eye? Glaucoma patients frequently exhibit abnormal Tcell subsets and increased titres of serum antibodies to retina and optic nerve proteins; these alterations in the cellular subset indicate that the immune system plays an important role in the initiation and/ or progression of glaucomatous optic neuropathy.48 Therefore, serum antibodies to retina and optic nerve proteins might be considered as potential indicators for diagnosis of glaucoma pathology.21 In
DNA Damage and Repair Capacity in Glaucoma Research work focused on the ex vivo comparative investigations of DNA damage in circulating leukocytes isolated from patients with glaucoma demonstrated significantly enhanced DNA damage compared to both healthy vasospastic and nonvasospastic individuals.28 Comparative comet assay analysis revealed patterns of comets typical for glaucoma patients as shown in Fig. 1. These findings suggest comet assay profiling of DNA damage in circulating leukocytes as a potentially powerful tool for noninvasive molecular diagnostics of glaucoma disease. Comet assay analysis as a suitable tool for biomarkers has also been suggested for Alzheimer disease.27 Comet assay analysis reveals enhanced DNA damage in both high- and normal-tension glaucoma.28 Whether the level of DNA-damage correlates with disease severity remains to be clarified. Further studies should also evaluate whether significant increase in DNA damage of leukocytes of glaucoma patients is caused by either disease specific stress factors, such as local ischemic/ reperfusion events, and/or decreased capacity of DNA-repair machinery. There is some evidence for
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STRESS RESPONSE, APOPTOSIS AND DNA REPAIR
An increased oxidative stress has been well documented under glaucoma pathology. Rat glaucoma models have demonstrated numerous oxidatively modified proteins in retinal protein lysates from ocular hypertensive eyes.42 Consequently altered gene expression patterns, particularly, for stress response factors can be expected. Thus, heat shock 27 kDa protein 1 demonstrated stable expression alterations in retina hypertensive eyes of rat.1 Gene hunting technology carried out in circulating leukocytes of glaucoma patients revealed an up-regulation of both the pro-apoptotic factor p53 and proteolytic enzyme 20S proteasome subunit XAPC7, activity of which is usually increased during reperfusion.18,47 In consensus, both an apoptotic inhibitor surviving and DNA-repair Xeroderma pigmentosum gene C have been found to be downregulated in circulating leukocytes of glaucoma patients.18 ADHESION, BLOOD--BRAIN BARRIER BREAKDOWN, AND TISSUE REMODELING
Fig. 1. Comet assay ex vivo analysis of DNA damage in circulating leukocytes. Top: Comet patterns typical for healthy controls: there are no visible tails and the majority of chromosomal DNA is focused in heads of comets (intact DNA). Bottom: Comet patterns typical for glaucoma patients: in contrast to the control patterns in the figure on the left, there are clearly visible tails (damaged DNA) and heads of comets are diffuse.
both eventualities: simultaneous up-regulation of p53 (stress regulated gene) and down-regulation of XPGC (essential member of DNA-repair machinery) has been ex vivo demonstrated in circulating leukocytes of glaucoma patients18 and represent potential molecular blood markers for the disease.
Key Molecular Pathways Affected by Glaucoma Pathology: Development of Potential Disease Specific Biomarkers The majority of gene products, of which the expressions are altered in glaucomatous pathology, can be well-organized functional groups belonging to distinct molecular pathways as described subsequently.
Gene expression profiles characteristic for an activated adherent function have been identified in circulating leukocytes of glaucoma patients: upregulated transcripts of lymphocyte-IgE-receptor (Fc-epsilon-RII/CD23), T-cell-specific tyrosine kinase (ITK), thromboxan-A2-receptor, and alkalinephosphatase have been detected by subtractive hybridization.15 Adherent circulating leukocytes could be an important contributor to blood--brain barrier (BBB) breakdown observed in glaucoma pathology.20 The subtraction of metalloproteinase MMP-9 and MT1-MMP transcripts highly increased in circulating leukocytes of glaucoma patients provides further evidence for this functional link and indicates pathways involved in extensive tissue remodeling observed in glaucoma.16 Significantly increased protein expression rates of both latent and active forms of MMP-9 and MT1-MMP in circulating leukocytes correlated well with the enhanced levels of transcription and with glaucoma.16 Once activated, both hydrolyses necessarily contribute to remodeling or even degeneration of the tissue whereto they are secreted by circulating leukocytes. This up-regulation might be a consequence of repeated mild ischemia/reperfusion postulated in glaucoma patients. However, the question of whether there is a correlation between increased metaloproteinase (MMP) activity and glaucoma severity should be further clarified. Recently performed comparative gene transcription profiling in trabecular meshwork isolated from postmortem glaucomatous eyes revealed a significant
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up-regulation of several cell adhesion molecules, including platelet endothelial cells adhesion molecule-1 and P-selectin.9 An enhanced deposition of the cell aggregation protein cochlin in trabecular meshwork and Schlemm’s canal of glaucomatous eye4 supports the crucial role of cellular adhesion in glaucoma pathology and favors cell adhesion molecules as potential disease biomarkers. TRANSCRIPTION REGULATION
Dramatically altered transcription patterns in glaucoma were reported by several research groups.1,15,16,18 A reason for this extensive shift in transcription regulation was unclear for a very long time. Very recently a significant up-regulation of the basic transcriptional regulator AP-2ß was attributed to glaucoma pathology: AP-2ß was identified in circulating leukocytes using the smart technology of disease proteomics.17 Protein mapping for circulating leukocytes is demonstrated in Fig. 2. AP-2 proteins play a decisive role, particularly, in morphogenesis of eye. While the activation of AP-1 leads to increased stromelysin (metalloproteinase-3) production in trabecular meschwork cell cultures,13,31 the expression and activity of AP-2 control the activity of the gelatinase B (MMP-9).29 MMP-9 was demonstrated to be highly activated in leukocytes of glaucoma patients and plays an important role in glaucoma pathology.16 Therefore, the conserved up-regulation of AP-2ß in leukocytes of glaucoma patients has been proposed to be an important part of molecular mechanisms of glaucoma pathology.
Fig. 2. Ex vivo protein mapping by 2D-PAGE imaging in circulating leukocytes of glaucoma patient. For this image, silver staining was performed. On average about 10,000 proteins are supposed to be simultaneously expressed in human leukocytes. Currently most sensitive silver staining visualizes not more than 300 spots for further qualitative and quantitative analysis of protein expression patterns.
GOLUBNITSCHAJA AND FLAMMER MULTI-DRUG RESISTANCE
An extensive dysregulation of ABC-transporters has been demonstrated in glaucoma pathology.10,18,49 ABC-transporters (ATP-binding cassette transporters) usually translocate a wide variety of structurally unrelated lipophilic compounds being responsible for drug efflux, and therefore, for multidrug resistance. ABC 1 has been shown to be stably up-regulated in circulating leukocytes of glaucoma patients.49 Joyce et al demonstrated a crucial role of ABC 1 in protection against atherosclerosis.23 An activity of ABC 1 has been shown to have a regulating effect on the endothelial function and stimulate nitric oxide bio-activity in arterial walls.5 The upregulation of ABC 1 in circulating leukocytes of glaucoma patients might indicate the involvement of this gene in chronic vascular dysregulation frequently observed in glaucoma pathology, and has been suggested as a potential marker for early diagnostics of glaucoma.49 ENERGY METABOLISM
Gene hunting in circulating leukocytes of glaucoma patients has revealed down-regulated transcripts of Na þ/Kþ-ATPase.15 An identification of the subtracted transcripts is demonstrated in Fig. 3. Further, the abnormal sodium handling has been proposed to be associated with ocular hypertensives and to contribute to a progression of the optic nerve damage in both normal-tension and high-tension glaucomas.32,36 Naþ/Kþ-ATPase is known to be down-regulated in lymphocytes of patients with acute myocardial infarction,34 and may be one of the reasons for ventricular arrhythmias and coronary artery spasms. Decrease of intracellular potassium concentration and increase of intracellular calcium concentration may play a major role in the
Fig. 3. Subtractive hybridization is one of the most powerful technologies of gene hunting strategies. Here the subtracted sequence (subtr.) is aligned demonstrating 100% homology to human Naþ/Kþ-ATPase. The subtracted sequence was found to be significantly downregulated in circulating leukocytes of glaucoma patients compared to controls.
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Possible Types of Quantitative and Qualitative Molecular Alterations in Glaucoma Type of Molecule Chromosomal DNA, mitochondrial DNA mRNA
Proteins
Metabolites (signaling molecules, amino acids, plasma hormones, etc.)
Possible Type of Alteration 1. 2. 3. 4. 1.
(Oxidative) damage* Mutations* Polymorphism* Methylation status of CpG islands** Multiple alterations in expression patterns*
2. Reduced mRNA editing 1. Multiple alterations in expression patterns* 2. Posttranslational modification* 3. Phosphorylation status* 4. Protein misfolding* Altered profiles*
Detection Technology -
Comet assay Disease genomics PCR, Restriction analysis, etc. Methylation-specific PCR Disease transcriptomics: Subtractive hybridization, Expression array, ReverseTranscriptase-PCR, Real-TimePCR, etc. - Reverse-Transcriptase-PCR - Disease Proteomics: 2D-PAGE, MALDI-TOF, Western-blot, etc. - Western-blot, Activity tests (e.g. Zymography for gelatinases) - Activity tests - Activity tests Disease Metabolomics: Comparative blood plasma metabolites profiling, high performance liquid chromatography, Activity tests, etc.
MALDI-TOF5matrix assisted laser desorption/ionization time-of-flight; PCR5polymerase chain reaction. Types of molecular alterations reported for glaucoma pathology. ** Data indicating this kind of alteration collected in our laboratory, which have not been published until now. *
pathomechanism of coronary artery spasms. Inhibition of Naþ/Kþ-ATPase is one of functional consequences of oxidative membrane damage caused by an increased oxidative stress,40 which is triggered, for example, under ischemia/reperfusion. This affects central thermogenic mechanisms, because Naþ/Kþ-ATPase has been shown to play a key role in cellular energy balance and thermogenesis.37,44 A decreased activity of Naþ/Kþ-ATPase triggers vasospasm.2 Being an important co-factor of cellular ATPases, magnesium can reverse delayed vasospasm and reduce the extent of acute ischemic lesions.45 Magnesium therapy demonstrates beneficial effects in vasospastic syndrome and glaucoma.12
Effect of Glaucoma Medication on the Evaluation of Biomarkers Evaluation of glaucoma biomarkers under treatment conditions is a particular issue in glaucoma research. Indeed, can we use the markers proposed for diagnostics of untreated glaucoma also under glaucoma medication? Are there any reliable markers that can predict the outcome of glaucoma treatment? There are some studies clearly demonstrating that glaucoma medication may definitely mask alterations in crucial biomarkers such as those of the nitric oxide pathway.25 Additionally, toxic side effects of anti-glaucoma drugs on conjunctiva have been reported: histological studies of conjunctival
tissues in patients who underwent a long-term glaucoma treatment demonstrated an abnormal infiltration of inflammatory cells. Consequently, an abnormal expression of inflammatory markers under various anti-glaucomatous treatments has been shown.3 Much progress has been achieved in the development of markers, which can potentially predict the outcome of glaucoma surgery. In the research work done by Souchier et al, surgery success was defined as dropped IOP # 15 mm Hg without any IOP-lowering drugs at 6 months, and conjunctival expression levels of trefoil factor family 1 (TFF1) and MUC5AC as well as HLA-DR in leukocytes have been measured.38 An increased expression of all three molecular markers has been proposed to be a potential predicting factor of successful glaucoma surgery. However, the issue of glaucoma medication markers is a multifarious one and requires more research effort.
Perspectives for Application of Most Potent Biotechnological Tools High-accuracy proteome maps of human body fluids open new perspectives for early molecular diagnostics of chronic disorders. Thus, recent identification of 491 proteins in the tear fluid proteome reveals a large number of proteases and protease inhibitors.8 An unbalanced secretion of
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proteases against their inhibitors might play an important role in pathogenesis of glaucoma as demonstrated, particularly, for MMP-9 up-regulated in blood.16 Until now, tear fluid has not been investigated for the presence of specific biomarkers in glaucoma. However, proteome research clearly demonstrated that tear fluid should generally be considered as an important source for biomarkers. Obviously this is a potential source for glaucoma biomarkers as well. The human differential blood proteome holds the promise of a revolution in epidemiologic screenings, disease diagnostics, and therapeutic monitoring provided major challenges in proteomics and related disciplines can be addressed also in oncology. Altogether about 3,000 and 10,000 protein products have been reported to be usually present in human plasma and circulating leukocytes, respectively. Abundant scientific evidence from disease proteomics suggest that disease-specific protein expression patterns in blood are indicative for many—if not most—human disorders, and might be a powerful tool for early molecular diagnostics of glaucomatous damage. A non-invasive molecular diagnostic approach based on disease specific gene expression patterns in circulating leukocytes has been recently suggested for glaucoma (Golubnitschaja and Flammer, International Patent No. IB02/00648). The test foresees a precise expression profiling of selected genes in circulating leukocytes isolated from fresh blood samples. These genes have been proposed to play a role in glaucoma pathology and belong to following pathways: apoptosis, stress response, the DNA-repair, transcription regulation, drug resistance, tissue remodeling, and degeneration. Currently a clinical application of the test is under consideration, and a nanotechnology, which should provide a possibly easy and cheap routine application of the test, is under development. Possible types of molecular alterations that can be considered for further development of glaucoma markers as well as corresponding detection technologies are demonstrated in Table 1.
circulating leukocytes. No specific exclusion criteria were used. No time limitation was set, and the following years are covered: 1992--2007. Although no language preference was intended, only articles in English were found and used.
Method of Literature Search The literature search was performed in PubMed. The following search words were used: glaucoma and molecular diagnosis, neurodegeneration, DNA damage/ repair/methylation status, gene hunting, disease transcriptomics and proteomics; proteomes of human body fluids; gene expression patterns in glaucoma, glaucoma medication; glaucoma and bio-markers, stress response, apoptosis, DNA-repair, cell adhesion, tissue remodeling, multi-drug resistance, energy metabolism, blood proteome,
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S161 35. Schmutzler RK, Rhiem K, Breuer P, et al: Outcome of a structured surveillance programme in women with a familial predisposition for breast cancer. Eur J Cancer Prev 15:483--9, 2006 36. Schwartz B, Abrahamson R, Takamoto T, et al: Association of ocular pressure and optic disc cup volume with red blood cell sodium-potassium ATPase inhibition. Curr Eye Res 21: 897--905, 2000 37. Silva JE: Thyroid hormone control of thermogenesis and energy balance. Thyroid 5:481--92, 1995 38. Souchier M, Buron N, Lafontaine PO, et al: Trefoil factor family 1, MUC5AC and human leucocyte antigen-DR expression by conjunctival cells in patients with glaucoma treated with chronic drugs: could these markers predict the success of glaucoma surgery? Br J Ophthalmol 90:1366--9, 2006 39. Sripriya S, Uthra S, Sangeetha R, et al: Low frequency of myocilin mutations in Indian primary open-angle glaucoma patients. Clin Genet 65:333--7, 2004 40. Stark G: Functional consequences of oxidative membrane damage. J Membr Biol 205:1--16, 2005 41. Tatton W, Chen D, Chalmers-Redman R, et al: Hypothesis for a common basis for neuroprotection in glaucoma and Alzheimer’s disease: anti-apoptosis by alpha-2-adrenergic receptor activation. Surv Ophthalmol 48(Suppl 1):S25--37, 2003 42. Tezel G, Yang X, Cai J: Proteomic identification of oxidatively modified retinal proteins in a chronic pressureinduced rat model of glaucoma. Invest Ophthalmol Vis Sci 46:3177--87, 2005 43. Tomarev SI, Wistow G, Raymond V, et al: Gene expression profile of the human trabecular meshwork: NEIBank sequence tag analysis. Invest Ophthalmol Vis Sci 44:2588--96, 2003 44. Valdemarsson S, Ikomi-Kumm J, Monti M: Increased lymphocyte thermogenesis in hyperthyroid patients. Role of Na/K pump function. Evaluation of aerobic/anaerobic metabolism. Acta Endocrinol (Copenh) 126:291--5, 1992 45. van den Bergh WM, Dijkhuizen RM, Rinkel GJ: Potentials of magnesium treatment in subarachnoid haemorrhage. Magnes Res 17:301--13, 2004 46. Wiggs JL, Auguste J, Allingham RR, et al: Lack of association of mutations in optineurin with disease in patients with adult-onset primary open-angle glaucoma. Arch Ophthalmol 121. 118--3, 2003 47. Wunderlich K, Golubnitschaja O, Pache M, et al: Increased plasma levels of 20S proteasome alpha-subunit in glaucoma patients: an observational pilot study. Mol Vis 8:431-5, 2002 48. Yang J, Patil RV, Yu H, et al: T cell subsets and sIL-2R/IL-2 levels in patients with glaucoma. Am J Ophthalmol 131:421-6, 2001 49. Yeghiazaryan K, Flammer J, Wunderlich K, et al: An enhanced expression of ABC 1 transporter in circulating leukocytes as a potential molecular marker for the diagnostics of glaucoma. Amino Acids 28:207--11, 2005 The authors reported no proprietary or commercial interest in any product mentioned or concept discussed in this article. Reprint address: O. Golubnitschaja, PhD, Professor and Head of the Division of Molecular/Experimental Radiology, Radiological Hospital, University of Bonn, Sigmund-Freud-Str. 25, D-53105 Bonn, Germany.