NATURE VS NURTURE:
OLD CONTROVERSY AND NEW THOUGHTS FOR GLAUCOMA

Jon R. Polansky, MD, UCSF Medical Center

correspondence:
Jon R. Polansky, MD
Department of Ophthalmology, Room K-301
UCSF School of Medicine
San Francisco, CA 94143-0730

George Spaeth: “I was hoping you would discuss how it is that nurture effects nature. That is, how does, the environment effect gene expression, etc. How do topical steroids effect trabecular function by changing the expression of gene? Why do fraternal twins and happily married couples have similar cycles of corticosteroids, etc etc. Let me have your thoughts.”

These and other sources of evidence have sought to implicate hormonal (potentially estrogen/ progesterone, androgen, thyroid, and corticosteroid) and injury (lipid peroxide/ hydrogen peroxide, and possibly oxidized pigment membranes) mechanisms in glaucoma pathogenesis. By framing the question as being one of “nature vs. nurture,” Dr. Spaeth is asking me to use this structure to discuss some of my current thoughts concerning environmental-genetic influences, including recent clinical and basic evidence from our work on the TIGR (aka myocilin, MYOC, TIGR/MYOC) gene. I do so with the understanding that this subject is just at its initial phases of both our knowledge and the probable several applications that will come from such an approach. As stated by centuries earlier by the poet, “What immortal hand or eye could frame thy fearful symmetry?” -William Blake, “The Tyger”

I. Discovery of the TIGR/MYOC Gene in Relation to Glaucoma:

The discovery of the TIGR/MYOC gene in relation to glaucoma depended upon both classical genetics gene expression approaches. Efforts are underway to follow-up on TIGR /MYOC gene regulation studies in human trabecular meshwork (HTM) cells (and other possibly relevant cell types), in addition to ongoing mutation studies, because of potentially important clinical implications..

Coding region mutations: TIGR /MYOC (or MYOC) became generally recognized as an important gene that was involved in glaucoma pathogenesis beginning approximately six years ago (1-3). The finding became the major impetus for widespread interest in the gene. Screening of mutations and variants/ polymorphisms was facilitated by the publication of the complete sequence of the TIGR/MYOC gene, including its promoter, introns, and exons, by Nguyen et al.(3). Over the past several years, many groups have confirmed the initial report by Stone et al.(1) of probable disease causing mutations in the coding region of the gene associated with glaucoma (e.g. see 4-11). Disease associated point mutations are often found to be in the third exon of TIGR /MYOC, and are predicted to exert a substantial influence on protein structure. While there has been speculation as to the mechanisms involved in the pathogenic effects for a number of the mutations, the processes leading to the development of glaucoma involving TIGR /MYOC remain to be elucidated.

Cloning of TIGR/MYOC from HTM cell models of gene regulation in glaucoma: We had originally used TIGR /MYOC gene regulation by corticosteroids (glucocorticoids, GCs) to characterize the HTM cell model for steroid glaucoma, and found the same gene to be induced by oxidative stress/ injury in our POAG cell culture model (2, 12, 13). When the gene was found to be within the region of chromosome 1 for the ‘GLC1A locus’ (2, 12), it became apparent that mutations in TIGR/MYOC were likely to be responsible for the linkage observed in familial juvenile glaucoma determined by other groups (14). The observation that the TIGR/MYOC expression appears to be increased in a majority of specimens evaluated using antibody made from rTIGR protein (15), and continued work on hormonal and stress-regulation of gene expression (16, 17), have expanded our understanding of how TIGR/MYOC could be involved in glaucoma pathogenesis. The finding that normal TIGR /MYOC protein may contribute to outflow obstruction in an anterior segment perfusion model (18) supports the idea of physiological as well as pathological effects of changes in the protein production, removal, and/or binding The work on TIGR/MYOC regulation is providing potentially important conceptual mechanisms by which the gene can be involved in glaucoma in cases where disease-causing mutations are not present.

In the presentation, ideas concerning environmental-genetic interactions are presented in relation to TIGR/MYOC studies and further applications of the HTM cell model system are updated from certain recent information published previously (see 12, 13, 17, 19). Current findings that appear to provide potentially useful leads will also be presented, but there will not be sufficient time to fully consider all of the data and references that might bear on the subject. Interestingly, in evaluating the HTM cell model for genes co-regulated with the TIGR/MYOC gene in HTM cells, our recent findings have implicated certain Alzheimer disease (AD)-related proteins (20, 21), that appear to be responding to corticosteroid and other types of stress as “acute phase response” proteins that could play a role in glaucoma. Other findings appear to implicate “environmentally available” hormones and endogenous growth factors as proving a balance for endogenous stress factors in relation to TIGR/MYOC and glaucoma pathogenesis. On the interface between the environment and genetics, a change in the TIGR/MYOC gene promoter region (termed the mt.1(+) variant [-1000 C/T]).

II. Genetic Evidence from studies of TIGR/MYOC mutations

The evidence supports the view that individual point mutations of TIGR /MYOC are likely to be causally related to the development of glaucoma in many of the families with juvenile-onset disease, and mutations of the gene are expected to be found in approximately 2-4% of adult OAG cases (e.g. 1, 5, 9, 23). In addition, it appears that the likelihood of an individual adult OAG patient having a coding region mutation appears to be substantially increased if one has elicited a positive history of a family member who developed glaucoma before the age of 35 years old (10). Certain mutations appear to be associated with a more aggressive and an earlier onset form of glaucoma (e.g. Pro370Leu). The relatively common Gln368Stop is associated with a milder, later onset form of OAG, and a more variable inheritance pattern (1,5, 10, 23, 24). Alward et al. (23) have emphasized that 396Ins397, Tyr437His, and Ile477Asn also appear to be associated with a more aggressive form of glaucoma, and it is possible individuals with these mutations may be more resistant to medical and laser treatment than might otherwise be expected. Improved methods to investigate the effects of medications and other interventions on the disease course are needed, including the possible use of time-to-event analyses of glaucoma progression using measures of disc and /or field changes, to assess this and other effects.

Evaluations of the different TIGR/MYOC mutations holds the prospects of understanding potential pathogenic mechanisms in glaucoma. As one potentially useful approach to grouping TIGR/MYOC mutations, Rozsa et al.(25) have provided analyses of various coding region changes from their work and that in the literature. Among the observations made by the authors, is a potentially important clustering of coding region defects in certain parts of TIGR/MYOC, and especially in the gene’s region of olfactomedin (OLF) homology of exon 3 (a possibility that includes sites of possible phosphorylation, as a predicted area of interest). The results can provide a framework to examine potential functional changes in the abnormal TIGR/MYOC proteins being evaluated in model systems. Such information should be helpful in designing and/or interpreting studies involving the expression of the abnormal gene.

The major OLF domain present in TIGR/MYOC’s third exon could have an important functional role, and may be an area that has developed stringent ‘quality control’ mechanisms for protein biogenesis in the secretory pathway. The OLF family of proteins rather than myocin shows the greatest homology to TIGR/MYOC protein (3), and olfactmedins include other extracellular glycosylated forms. We have previously emphasized that the extracellular expression of TIGR/MYOC was likely to be one crucial aspect of the molecule’s effect on outflow resistance in glaucoma (2,3)..TIGR/MYOC coding region mutations affecting the gene’s OLF homology domain may perturb biosynthetic pathways and cellular homeostatic functions, both inside and outside of the cell. In this regard, our HTM cell transfection studies using TIGR-green fluorescent protein (GFP) fusions showed increased and altered distribution of the expressed protein with constructs missing the OLF domain, an effect also found with the Pro370Leu mutation for early-onset glaucoma (16). Continued evaluations of normal and altered biogenic pathways concerned with TIGR/MYOC variants/ mutations, should keep in mind the possibility that an activation of stress/apoptotic pathways in HTM cells could be taking place as a potential mechanism for environmental/ genetic interactions in glaucoma pathogenesis.

Evidence is being uncovered that supports the idea that individual pathogenic mechanisms may be playing a role in how various TIGR/MYOC mutations exert their effects. In one example, a collaborative study of our laboratory and that of Lingappa has shown that there are some mutations (e.g. Glu323Lys) that appear to be influencing what is termed a ‘translational pause’ in the protein’s biogenesis (26). This and other nearby mutations have been found that have little predicted structural changes in the expressed protein based on the sequence change. The observation that Glu323Lys and other mutations in the adjoining region alter the pattern of paused protein intermediates provides possible clues to previously unexplained pathogenic mechanisms. In another example, an extensive family shows homozygotes for the Lys423Glu mutation that are unaffected, but has heterzygotes that are affected (27). The primary explanation given by the main authors involves a postulated “homoallelic complementation” mechanism, but other explanations appeared possible when considering the evidence previously (12). Another possibility, consistent with the clinical observations and experimental studies that have been more recently conducted, is that abnormal gene products may be accumulating in the HTM cell, producing a stress response and an increased amount of extracellular TIGR/MYOC protein that might contribute to outflow obstruction. Also, in line with such a concept, is the finding that homozygotes carrying a TIGR/MYOC sequence change (Arg46Stop) allowing for only a small truncated molecule to be expressed, show no glaucoma association (28). This observation reinforces ideas made in clinical evaluations of haploinsufficiency without glaucoma (29), as well as more recent transgenic experimental studies by Tomarev’s group (30) that favored the idea of “a gain in function” rather than a loss of function with TIGR/MYOC mutations.

As one aspect of environmental/ genetic interactions, the clinical effects of disease-associated sequence changes in the gene might be influenced in substantial ways by ‘modifier genes’ and/or endogenous factors that coul alter pathogenic processes affected by the mutations. This idea was recognized in papers by Garchon (31), and also by my laboratory (13,19). Such effects could potentially explain the observed spread of ages in which onset occurs due to the same TIGR /MYOC mutation. It is also possible that useful clinical information might be gained if an appropriate evaluation of the onset or clinical features of glaucoma in OAG subjects with the 368Stop mutation would be conducted, for example, with regards to those individuals with the mt.1(+) or other TIGR/MYOC variants.

The TIGR/MYOC mt.1(+) variant and glaucoma severity/ progression

In a recent paper we have reported a strong association of the mt.1(+) variant with an increased risk for more rapid glaucoma disease progression, and have proposed its role in glaucoma management (22). In addition, since the genetic variant is inherited, it might provide a potential early warning for certain populations and situations, an idea that needs to be evaluated further.

Our initial attention was drawn to the mt.1(+) variant several years ago, since it appeared to be the only one of several promoter variants we screened that was present in a greater percentage of those with the disease than in controls. Evaluations we were conducting with other investigators suggested to that the finding appeared to be valid only for the more severe glaucoma cases. Independently, a multi-variant approach of the glaucoma phenotype was employed by Colomb et al. (32) from Garchon’s group for the mt.1(+) variant, reaching a similr conclusion for an association of visual filed loss with the mt.1(+) variant, with the added complication that there was an IOP effect involving both mt.1(+) and ApoE variants (the effect on the visual field appeared to remain when double mutants involving ApoE variants associated with resistance to IOP lowering were removed from the analyses - Garchon 2003, personal communication). Other studies of the glaucoma phenotype seen in mt.1(+) patients, appeared to show an association with greater disease severity using a 4 point scale to assess optic disc changes - but neither this study nor the prior ones permitted a proper assessment of changes as a function of time, nor an assessment of the role of other glaucoma risk factors. It was, therefore, desirable to conduct more definitive studies of the possible association of mt.1(+) with worsening of glaucomatous disease over time. The findings of an accelerated worsening of both disc and field measures of glaucomatous disease were recently published in a study conducted with Dr. George Spaeth at the Wills Eye Hospital (22).

The study with Dr. Spaeth found an association of the mt.1(+) variant and disease severity/ progression. The association was examined relative to other risk factors for glaucoma in a time-dependent analyses of 147 patients followed for an average of 15 years. The value of assessing ‘risk characteristics’ for developing glaucoma in the context of the analysis of the risk of glaucomatous progression emphasized the expected multi-factorial influences over the glaucomatous disease process. Of substantial interest was the observation that the association of the variant with accelerated disease progression was found to be robust even when the other strong effect of age was taken into account. This was true for both the optic disc data and the visual field data, analyzed as separate effects. Using time-to-event Cox model statistical analyses, mt.1(+), age, original severity measure, family history, and diabetes (protective effect) were found to be significant risks involved in the measures of progression. In the hazards model, for both the optic disc analysis and the visual field analysis, mt.1(+) and age were the two largest effects, and both showed a clear statistical association in the optic disc and visual field analyses.

Figure 1 and Table I, and Figure 2 and Table II, show the results of the Cox model analyses from our published paper, in which a 55 year old person at baseline was selected to illustrate the difference between the mt.1(+) vs mt.1(-) genotypes. The risk of a two-step change in optic disc or visual field measures of progression was evaluated over extended observation times, as indicated on the figures (for the analyses shown, the values for other risk factors generally set to group averages, according to the descriptions in the paper). A two-step change that was selected in order to minimize ‘noise’ due to reversals, helping ensure that actual disease progression had taking place. In the figures, the top solid line represents the mt.1(+) findings (90% confidence intervals shown by dotted lines); the bottom solid line represents the mt.1(-) findings (with 90% confidence intervals indicated). As the examples selected illustrate in the figures and tables, there is a substantially greater risk of progressing with optic disc or visual field damage if a patient is mt.1(+). The follow-up evaluations showed that the risk for progression in the optic disc was much less in mt.1(-) patients (6% and 18%, at 10 and 15 years, respectively) vs mt.1(+) patients (55% and 93%, at 10 and 15 years, respectively). The visual field data show similar associations: with mt.1(-) patients showing 12% and 33% (at 10 and 15 years, respectively) vs mt.1(+) showing 58% and 93% (at 10 and 15 years, respectively).

The fact that the TIGR/MYOC mt.1(+) variant is commonly found in 15-20% of individuals tested, suggests that a substantial part of one’s glaucoma practice may be at greater risk than has been generally appreciated. Importantly, our analyses of the risk associated with the mt.1(+) genotype for accelerated disease progression did take into account the ‘time-to-event’ changes in the optic disc and visual field data, as well as other potential risk factor for disease progression, with the association of the rate of progression showing the largest effects with mt.1(+) and with age. . Interestingly, one may not readily see a clear effect on a single baseline visit without having sufficient follow-up and risk factor data. For example, Alward et al. (23) had observed that the mt.1(+) variant could not be used to distinguish his more severe and less severe phenotypes coming to the Iowa clinics, and this finding is in agreement with our baseline data that does not consider time dependent changes (progression) and the effects of other glaucoma risk factors. This information supports the potential effect of the mt.1(+) genotype, even in the context of careful monitoring of patients in a specialized glaucoma clinic.

TIGR/MYOC gene regulation

Our studies of HTM cell specific gene regulation led to the cloning of TIGR/MYOC as a putative new glaucoma gene, and also is providing leads to clinical glaucoma mechanisms whether there are mutations present or not in TIGR/MYOC. Morphologically differentiated HTM cells grown in culture were used to look for the effects of GC and other ‘stress’ treatments as model systems to study glaucoma mechanisms. We postulated that use of stress-injury treatments (for which there were suggested glaucoma mechanisms), would allow us to ‘perturb’ the gene expression in differentiated HTM cells in unique ways, permitting an identification of molecular species that could play roles in steroid glaucoma and/or POAG. The observed specificity in the response to chronic dexamethsone (DEX) exposure, including very high induction levels and extracellular expression in HTM cells compared to other cell types examined (2,3,16), seemed to support our approach. The first application led to the identification of the novel gene product, that later became known as TIGR/MYOC. Our cloning of TIGR/MYOC cDNA revealed structures of substantial interest in both the coding a non-coding regions of the gene. In addition to the OLF domain mentioned earlier, and other noteworthy structural features we will not review here, the gene’s putative promoter region contained several potential hormonal and other regulatory motifs (partial nGREs, Ap-1, TRE’s and others), as described (2,3). Ways in which the TIGR/MYOC gene and the expression of its products in different cell types may be regulated by “environmental” influences, as well as by sequences in the genetic code, are areas of substantial interest to us in relation to glaucoma mechanisms. Studies of TIGR/MYOC gene promoter functions, in the author’s opinion, will need to be conducted in relatively stable gene transfer studies using differentiated HTM cells to understand the major cell specific effects on the gene’s induction by GC and other stimulators/ modulators - an area currently proving to be challenging.

Evaluations of modulators of GC-induced TIGR/MYOC gene expression in the HTM cell model are providing additional information concerning potentially important “environmental” influences (16). For example, the DEX-induced increase of TIGR expression in HTM cells is reduced approximately 4-fold by basic fibroblast growth factor (bFGF, 100-1000 pM), with a somewhat smaller inhibition occurring with thyroid hormone, triiodothyronine (T3, 100 nM) treatment. It is possible that bFGF (and other endogenous growth factors) as well as triiodothyronine could act as ‘counterbalancing’ regulators that ‘protect’ against overproduction of TIGR/MYOC by a variety of stimulators. Potential stimulatory factors include TGF beta and mechanical perturbations as described by Tamm et al. (34) in addition to GCs and oxidative injury. Conceptually, one needs to consider whether a decrease in counterbalancing growth factors, thyroid hormones and/or other factors that might decrease with age (or in certain conditions) might contribute to outflow obstruction in POAG, especially if such unopposed stimulation would result in an increased TIGR/MYOC production in vivo. Basic FGF (i.e. FGF 2) and other growth factors are known to exert protective effects in other cell types, including those of the eye (35-37). The triiodothyronine effect is of additional interest because of the earlier proposals of an association between thyroid hormone deficiency and glaucoma, an idea that has received renewed attention in recent years (38, 39).

The observation that the HTM cell type appears to be particularly responsive with regards to stimulation of TIGR/MYOC production could have important ramifications, including providing an explanation for why gene defects, or why gene regulation/ modifier effects could be influencing the development or phenotype of glaucoma. In this regard, defects influencing translocation and/or translation (invoking ER sorting functions, degradative pathways and chaperone functions) may activate stress and/or apoptotic pathways which have a differential effect on HTM cells. The studies of Zhou and Vollrath (40) that used transfection of tagged TIGR/MYOC constructs into human kidney (HK) cells may be related to the processes being considered. The study involved evaluations of several TIGR/MYOC mutations in which an increased ration of the insoluble/soluble TIGR protein in detergent buffer was able to distinguish mutations from polymorphisms. Further studies may provide information regarding specific mechanisms in this interesting model system, some of which may be relevant to effects in HTM cells. In follow-up studies, it will be important to examine mutation/ polymorphism effects directly in HTM cells compared with HK and other cell types, under appropriate conditions to examine specific functions. In the studies, it is reasonable to expect there to be cell specific regulation found also involving ER factors (41) and perhaps other parts of the secretory pathway.

The possibility that abnormal forms of a protein could stimulate cell signaling pathways and the role for chaperone functions in the HTM could relate to studies of the ‘unfold protein response’ (UPR) in the secretory pathway. The UPR response involves the induction of cellular signaling pathways through an ER transmembrane protein with structural similarities to mammalian growth factor receptor kinases, along with the up-regulation of the transcription of ER resident genes involved in protein folding (41). This or a related cell signaling response to TIGR/MYOC mutations could provide a mechanism for our earlier proposal that some TIGR/MYOC mutations produce a stress induction of TIGR/MYOC proteins/glycoproteins in which both affected and normal genes could be influenced. As mentioned, these ideas appear compatible with the findings of B. Yue (personal communication, 2000) who found evidence for a mitochondrial form of TIGR/MYOC that also could provide part of the mechanisms involved in a stress-induced ‘apoptotic’ pathway activation with a marked GC-induced increase in the expression of TIGR/MYOC in HTM. Also compatible with a stimulation of apoptotic pathways in steroid glaucoma, Nguyen observed a noticeable elevation of bcl-2 in our 10 day, 500 nM DEX treated HTM cultures by RT-PCR (unpublished observations). If HTM cells respond to abnormal forms of TIGR/MYOC with an activation of ‘stress’ pathways, this would provide a means for alterations influencing intracellular or extracellular activities of the TIGR/MYOC species produced may play roles in certain of the disease-associated TIGR/MYOC mutations. Future studies of TIGR/MYOC expression in glaucoma will ideally consider a wide range of interactions, including effects on transacting factors that might influence TIGR/MYOC expression through the gene’s promoter region (or other regulatory sites) and effects on other genes whose products could interact with TIGR/MYOC protein/glycoprotein. The possibility that TIGR/MYOC may be induced in the optic nerve and other tissues exposed to stress in glaucoma also needs to be investigated further.

Continued Applications of HTM Model Systems

Use of GC-treated (and oxidative injury) HTM cell models appear to be an ongoing source of basic information, some of which may have direct clinical applications. As an example, it is possible that a subclass of potential interacting genes with TIGR/MYOC will help explain some recent clinical assocaitaions of glaucoma and Alzheimer’s disease (AD). Using our well-characterized HTM cell model systems, and searching for other candidate glaucoma molecules, Zimmerman et al (see 21, and unpublished data) have found a number of Alzheimer- and Acute Phase Response (AD/APR ) /stress-related proteins that appear to be co-regulated with TIGR/MYOC in the steroid glaucoma system. Certain of the species show major increases in gene expression in the steroid glaucoma HTM cell model that are different from other inductions in the same system, but parallel the induction profile of TIGR/MYOC, both in their timing and modulator effects, and in the differentiated systems that show the responses. Some of the genes of interest were also identified using microarray data conducted and evaluated in collaboration with Dr. Vollrath’s laboratory; others involved the concept of stress-related products discovered independently, with AACT being an example. Key findings were validated using of real-time reverse transcriptase polymerase chain reaction (RTPCR) studies of suspected AD/APR gene products. The studies have shown that certain genes, including alpha-1 antichymotrypsin (AACT), apolipoprotein D (APOD), serum amyloid A precursor protein (SAA1) and serum secretory leukoproteinase inhibitor (SLP1), had distinctive responses to GC treatments in HTM cells (and not other cell types) in that they showed a time course (and magnitude) for regulation, paralleling TIGR/MYOC in the steroid glaucoma model. Several other genes initially evaluated by microarry and proteomic approaches (some also evaluated by RTPCR), showed the more ‘normal’, expected (non-progressive and lower magnitude) GC inductions. Importantly, the RTPCR studies have shown that the AD/APR inductions could be observed clearly in confluent, differentiated HTM cells treated with prolonged DEX exposure, and were substantially less prominent-very low in growing HTM, or in growing or confluent DEX-treated skin fibroblasts. This ‘differentiation’ effect adds to the interest in the roles of these molecules in aqueous humor outflow pathway and in glaucoma. The inductions with DEX show a noticeable blunting when concommitantly treated using bFGF, similar to that seen with TIGR/MYOC. Studies designed to further examine AD/APR pathways in glaucoma pathogenesis appear of interest to elucidate how they may be related to stress responses of the HTM cells and other relevant ocular cells. It also appears that AACT may be interacting with TIGR/MYOC protein(s) within the secretory pathway, and may have a special role in relation to outflow obstruction (20).

Possibly related to our observations, a clinical study by Bayer et al (43) appeared suggesting an association of glaucoma AD and perhaps Parkinson's disease. Also, Garchon’s group (33) independently discovered a potential effect of APOE variants and the glaucoma phenotype seen with TIGR/MYOC mt.1, that may also provide leads to understanding how genes associated with AD may also play roles in glaucoma.

Summary and Perspectives

Ongoing studies of TIGR/MYOC are providing interesting leads to understanding the development of POAG and perhaps other forms of glaucoma. Defects in the gene’s coding region, and sequence variants in the gene’s promoter (e.g. mt.1), along with factors that could contribute increased environmental stress, are currently being assessed for their roles in disease pathogenesis and management. The assessments involved in this TIGR/MYOC gene research, will hopefully provide researchers with new leads for understanding glaucoma pathogenic mechanisms, and provide clinicians with practical information to allow a rational use of data being uncovered.

Continued efforts to define the pathways involved in normal and mutated forms of TIGR/MYOC could provide important clues into physiological/pathogenic mechanisms. In the coming years it will be of interest to determine the spectrum of effects produced by agents that both stimulate and reduce TIGR/MYOC mRNA. These studies are likely to be important in addition to direct measurement of effects on gene expression. Influences of chronic TIGR stimulation by GCs and other agents, in addition to the effects of TIGR/MYOC mutations on cell signaling and apoptotic pathways, need to be explored in much greater detail. Determinations of TIGR/MYOC’s ‘life cycle’ in different cellular compartments (including synthetic and degradative pathways produced by alterations in the molecule’s biogeneic pathways), may also help to explain certain pathological effects.

When mutations or variants of the gene are being considered, susceptibility and protective facts may be defined that influence the age of onset or observed clinical impact, in addition to effects of variants in the gene’s promoter and environmental/trans-acting factor influences that may function through the gene’s promoter, or other areas of the gene involved with regulation of expression.

Thus, in the clinical and basic evaluations being conducted, it will be important to take into account the spectrum of different genetic and environmental influences, i.e. the nature vs. nurture question(s) raised by Dr. Spaeth. Clearly there are many levels that such issues may come into play. Overall, the message is that we need to carefully consider in the design of clinical studies how different gene products could influence disease expression and the ‘clinical profile’ of an individual patient - rather than relying on an overly simplified genetic hypothesis.

Acknowledgments: The original work on cloning the TIGR/MYOC gene and its studies of its regulation were supported by NIH grants: EY02477 (to Dr. Polansky), EY08905 (to Dr. Thai Nguyen), and the Departmental Core grant, EY02162. The more recent findings reported here have been supported by the Glaucoma Research Foundation, That Man May See, Inc., and/or by InSite Vision, Inc. Dr. Polansky has a financial interest in the research presented.

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