Probing the genes behind the mechanics of glaucoma

ALTHOUGH glaucoma treatment has traditionally focused on the immediate mechanics of lowering raised intraocular pressure (IOP), ophthalmic researchers are now paying increasing attention to interfering with genes that may ultimately be responsible for the disease.

Most glaucoma genetics is premised on the theory that the disorder occurs because a blockage interrupts the balance between aqueous humour production and outflow.
With the block, IOP rises, subsequently leading to optic neuropathy and the death of retinal ganglion cells.

Researchers have long known that there is a hereditary component to glaucoma. For instance, the prevalence of primary open-angle glaucoma (POAG) in first-degree relatives of affected patients has been demonstrated to be seven to 10 times higher than that of the general population.

But from such an observation, how does a researcher begin to overcome what is essentially a needle-in-a-haystack challenge of locating the gene or genes at the root of the pathology?

Researchers have traditionally located ‘disease causing’ genes by applying one of two techniques: so-called ‘forward’ or ‘reverse’ genetics.
Forward genetics requires some prior knowledge of the biochemical defect that causes the disease pathology. For example, if one knows the protein that is involved in the pathology, determining the polypeptide sequence of the protein allows for the identification of the underlying genetic sequence and thus the identification of the gene and its location on one of the chromosomes.

However, in the absence of any biochemical clues associated with the pathology — such as is the case in glaucoma and virtually all retinal dystrophies — researchers must apply the principles of reverse genetics. Also known as ‘positional cloning’, reverse genetics attempts to localise a disease gene to a particular arm of a chromosome by tracking the pattern of what are known as ‘genetic markers’ in families that have the disease.

Genetic markers are recognisable landmarks found among the 3bn base sequences that represent the human genome. A genetic marker can help researchers locate a ‘disease’ gene by allowing them to observe the relationship between a given marker and the disease.

Researchers can then employ further refinements using molecular genetic techniques to locate the actual gene and mutation responsible for a given pathology.
Readers of this column may remember our discussion of the discovery of the “optineurin” gene by Mansoor Sarfarazi MD at the University of Connecticut Health Centre. Dr Safarazi reported that this new gene was associated with POAG in 16.7% of patients with the disease.

Studies are currently in place to determine the exact role of the optineurin gene; already, researchers have reported its presence within the trabecular meshwork indicating that it may be somehow involved in the biology of aqueous humour outflow.
Additional genes identified among the glaucomas include the myocilin gene mutated in juvenile open angle glaucoma (JOAG). Another gene is CYP1B1 (encoding cytochrome P4501B1 enzyme), which is mutated in chronic open-angle glaucoma (COAG). COAG is the late onset form of POAG,

Myocilin, also referred to as TIGR (trabecular meshwork-induced glucocorticoid response protein), has been shown to be expressed in the trabecular meshwork and ciliary body and is thought to cause an elevation in IOP by obstructing the outflow passages.

Recent studies of mouse models of glaucoma by Richard Libby MD and Simon WM John MD at the Jackson Lab in Bar Harbour, Maine, US, have identified a ‘modifier gene’ working in tandem with CYP1B1.
A modifier gene refers to a secondary gene responsible for the modulation of the expression of a specific genetic mutation and such a gene may be inherited independently of the primary gene responsible for the disease pathology.

In Dr Libby’s study, the modifier gene encoded an enzyme involved in the production of L-DOPA which has been used clinically to treat Parkinson’s disease. Consequently such findings open an exciting link to potential pathways involved in ocular development.
Such work readily illustrates the complexity inherent in disorders such as glaucoma which are thought to result not simply from a single defined mutation in a given gene but, rather represent a kaleidoscope of interactions between a primary gene or genes, a modifier gene or genes and environmental factors.

Variability in the age of onset, incomplete penetrance of the condition in some families and the prevalence of the disease all point to the involvement of more than one gene in addition to environmental factors affecting the expression of a particular genetic mutation.

Although each gene in isolation may logically follow Mendel’s laws of inheritance, the observed clinical heterogeneity derives from the potential number of genes involved and the added contribution of known and unknown factors.
POAG is thus considered a common but complex disorder and such complexity represents a significant challenge in fully characterising such disorders at the genetic level.
Nevertheless, the prevalence of the disease worldwide should ensure that extensive research will continue to illuminate the precise molecular genetic details behind POAG.

Of course the holy grail of all such studies is to reach a point at which knowledge of the underlying genetics allows for the development of therapeutic strategies focused on the genes involved. Decades of research into realising the potential of gene therapy define four key pre-requisites that should be met before any such approach is considered:

• An efficient and non-toxic gene delivery vector should be available
• The genetic basis of the disease should be well understood and characterised
• The therapeutic gene expression should be amenable to control
• Suitable animal models to test the therapies should be available

Glaucoma research is rapidly approaching the point at which these criteria may be met and as such, glaucoma may well become an attractive staging post for demonstrating the inherent power of gene therapy as a viable medical treatment.

Even without detailed genetic knowledge, there already exist gene therapy opportunities for the treatment of glaucoma. Genetic strategies may be directed at the specific cells and tissues of the anterior chamber that mediate the flow of aqueous humour in an effort to lower IOP. Of the genes identified to date, such as myocilin and optineurin, there may be several opportunities to deliver functional copies of these genes by viral vectors.

Furthermore, the end phase in the pathology of glaucoma has been shown to involve the apoptotic cell death of retinal ganglion cells. A significant body of literature now exists on the molecular biology of apoptotic cell death that may readily permit the introduction of anti-apoptotic gene constructs to inhibit the demise of these cells. Such a therapeutic strategy may well prove successful in halting or preventing vision loss.

Finally, in terms of commercial opportunity, the market profile of glaucoma represents an attractive investment should an appropriate technology present itself. With over 70m potential customers and no cure, the incentives to open up such a market will ensure a fiercely competitive environment. However, significant obstacles remain to challenge the budding biotech entrepreneur:
• Improved gene delivery techniques and vectors
• A better understanding of the molecular biology of aqueous humour production and outflow
• An improved understanding of the cell death pathways of retinal ganglion cells; improved control over gene expression
• A technology flexible enough to capture such a large market while at the same time addressing the underlying complexity and heterogeneity of the glaucomas
It’s a significant challenge for sure!

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