Ocular genetics advancing rapidly, but much more work remains

For the present, knowing the genes involved in a retinal disease may help in counseling patients.

Recent years have seen continuing growth in the quantity of genetic research and knowledge, and ocular genetics has been a large part of that research. It is now common for ophthalmic journals to contain articles discussing a newly discovered mutation or a study linking a gene to an ocular disease.

But gene researchers often say practical clinical applications, such as genetic therapies for inherited diseases, are still years away. Physicians want to know how ocular genetics today can assist them in clinical management of patients with genetic diseases.

In this article, Ocular Surgery News presents an overview of the state of ocular genetic research. This is not a gene-by-gene analysis of which diseases are mapped. Rather, it is a general look at what we know and how it may be of benefit to the clinician, now or in the near future.

Experts interviewed for this article said that currently, the clinician continues to play the crucial role in managing gene-related diseases. An accurate clinical diagnosis is necessary before mutation testing can be performed.

John R. Heckenlively, MD, a professor of ophthalmology at the Jules Stein Eye Institute and a researcher in ocular genetics, explained that there are many diseases influenced by genetics, and often more than one mutation may cause a given appearance. Researchers need an idea of where to start their search.

For example, roughly 84 monogenic types of retinitis pigmentosa have been described. All the variations originate from different genes, and recently this has been further complicated by the fact that one gene mutated in different spots can result in different expressions.

“The concept used to be that one gene equals one disease. And it turns out that concept is no longer valid. Depending on where the mutation is within a gene, you can get a different effect or a different disease,” Dr. Heckenlively said.

“You need some sort of idea what genes are involved. Then, after a good clinical diagnosis, it depends on the technique being used to track down the disease. but if it’s an unknown gene, one that hasn’t been discovered yet, then you have to start fresh,” he said.

Identifying a mutation

The identification of genes began with family studies, which demonstrated how a gene is inherited through generations. DNA would be extracted from individuals both affected and not affected by the mutation and compared against markers on chromosomes, which allow the identification of what chromosome carried the gene causing the disease.

Once a chromosome is identified, fine mapping can be done using microsatellite markers. This allows honing in more closely on the gene. Finally, sequencing and positional cloning techniques are used to pinpoint the mutation.

Much genetic research is now carried out in animal models. In particular the mouse can serve as a model because it is a mammal with an eye similar to the human eye. There is a 90% to 95% overlap in murine and human genes, Dr. Heckenlively said.

The retinas of both human and murine eyes have the same basic layers and structures. The mouse eye does not have a macula, and the lens is proportionally much larger than the human lens, but Dr. Heckenlively said these differences generally do not affect research.

“If we find a retinal degeneration in a mouse and identify the gene, there is about a 90% chance it will hold true in humans for causing the same retinal degeneration,” Dr. Heckenlively said.

The mouse is additionally useful because the species has a fast breeding time, allowing genes to be identified quickly. Mice also provide tissue with which to work toward gaining an understanding of the molecular and biochemical processes occurring in various degenerations.

Diagnostic testing

Research labs are also now better able to detect and diagnose a known mutation, which has led to a new problem.

“Now it is becoming almost a diagnostic test,” Dr. Heckenlively said. “Research labs are starting to wonder if performing these tests is still research. There is a movement to start charging patients to check whether a gene defect is the cause of their disease.”

He noted there has been slow movement in that direction, although no central facility has been established for widespread testing. A few commercial labs have made announcements indicating they are headed in such a direction.

Treatment approaches

Although research labs are better able to identify and diagnose mutations, therapies remain elusive.

To date, the most successful gene-based ocular therapy was performed by Jean Bennett, MD, PhD, and colleagues at the University of Pennsylvania. Their treatment was done on a group of dogs with an RPE-65 mutation, which causes Leber’s congenital amaurosis. This particular mutation causes the transport of vitamin A, or retinol, to be interrupted within the retinal pigment epithelium.

Patients with this condition do not immediately lose structural integrity of the photoreceptors, which remain intact sometimes for years before degenerating. By restoring a good gene for the pathway, Dr. Bennett’s group was able to restore normality to the metabolism of the photoreceptor layer.

There is hope this may be one of the first diseases in which gene therapy works in humans, said Birgit Lorenz, MD, a professor of ophthalmology and head of the department of pediatric ophthalmology, strabismology and ophthalmic genetics at the University of Regensburg, Germany. She noted that application in human retinal degenerations may not be as far off as some believe, with plans being made to begin human trials within the next 2 to 5 years.

Because the sequelae may be similar between defects, if gene-specific therapy is not feasible, another approach being considered involves targeting treatments at the sequelae or the functional and morphological consequences of the defect, Dr. Lorenz said.

“Let’s say five different gene defects lead to clinically similar disease. This may be mediated by growth factors. This may lead to a general therapy, which may not mean curing the disease but slowing the disease progress,” she said.

Dr. Lorenz said work with growth factors, like gene therapy, remains largely experimental.

How to target

According to Dr. Heckenlively, any genetics-based treatment would also depend on the inheritance pattern of the disease and what the gene does. The mechanism of action must be known in order to determine how to target the disease.

For this, several different approaches can be used depending on disease inheritance. For a dominant disease, it has been suggested that a ribozyme could be used to knock out either the bad gene or the RNA of the bad gene, which would leave the good gene to transport its RNA with the normal protein. He explained that in a dominant disease there is one gene for each pair of chromosomes and only one of the pair is defective.

“If you knock out the bad [gene], then you can let the good [gene] produce the protein. The approach depends on the type of genetic problem you have,” Dr. Heckenlively said.

Additionally, all known retinal degenerations follow the apoptosis pathway. The mechanisms of apoptosis, or programmed cell death, are becoming known, and the pathway itself has genetic mechanisms, he said.

“You may not cure it, but you can keep [the cell] from dying or at least delay it from dying,” he said.

Keeping current

Dr. Heckenlively said it will be increasingly difficult for clinicians to keep pace with advances of knowledge in genetics and move these discoveries into clinical practice.

“I think that we are going to have a period where science is going to be way ahead of clinical practice. It is going to be a challenge to implement these discoveries in a thoughtful and careful way,” he said. “In light of the financial restrictions we have, it is going to be even more difficult. I think we are going to be trying to cope with the flood of knowledge and yet have limited resources to deal with this new knowledge.”

Dr. Heckenlively explained that anytime a new therapy is brought forth, regulatory trials are required so the therapy can be labeled safe and effective. Each trial costs millions of dollars to run.

“Somehow we need to figure out how we can implement these [discoveries] without making the system expend millions of dollars,” he said. “Obviously you have to prove they are safe and effective, but is there a more effective way of doing it that is fiscally prudent? I don’t have an answer for that.”

Multiple genes

There are additional concerns when considering clinical trials of potential genetic therapies.

Dr. Lorenz said if a disease involves multiple genes, this would mean correcting each genetic defect separately. Adding to the problem, presently it remains unclear just how many genes are actually involved in eye diseases. A sufficiently large number of patients with the same gene defect would be required to do a therapeutic trial.

“The number changes, but let’s say there are 40,000 to 60,000 genes total. Probably at least 10% and likely more than 10% of all genes will be involved in the eye in one way or another. You can imagine the problems one would run into,” she said.

“Consider retinitis pigmentosa. This is a diagnosis that to the public is one disease, but there are more than 100 genes whose mutations all can cause RP, if you count the RP syndromes. If a therapy has to be gene-specific, this will be extremely expensive. It’s easy to get hundreds of patients to do a study [on diabetic retinopathy]. But to get hundreds of patients with a specific mutation in the rhodopsin gene, for example, would be very difficult,” Dr. Lorenz said.

“If you think about pediatric diseases, it would be almost impossible to get 100 young patients with the same gene mutation. Clinical trials will be complicated by this as well,” she said.

As diseases change over time, longitudinal studies are also needed, she said.

According to Dr. Lorenz, identifying genes and what diseases they code for is not a major problem for research clinicians because data banks and databases store such information. But because different diseases can appear clinically similar, one problem will be determining the origin of a disease that may be the result of different mutations in the same gene.

She again cited rhodopsin, explaining that it was one of the first genes identified as associated with retinal degeneration. Now, more than 100 different mutations of the gene have been described.

“The problem is that you cannot say by looking at a patient that he has retinitis pigmentosa associated with a rhodopsin mutation, unless he has sector RP when it becomes more likely. It becomes more and more difficult for the geneticist, clinician and molecular biologist to keep up with all the information. For the clinical data, it is much more complicated because the data are much more complex. As you can imagine, describing the disease and stating all the functional changes over time in a single patient is a vast quantity of data. It’s difficult to build up those databases,” Dr. Lorenz said.

“This is a major challenge for the clinician in this field, which brings us back to the problem of knowledge and the clinician,” she said.

Genetic counseling

Although treatments based on genetic research remain experimental, there is still immediate value in continuing the identification and classifying of genetic mutations and disorders, Dr. Lorenz said.

A real advantage in identifying and recognizing genetic origins for a disease, and knowing its inheritance pattern, lies in genetic counseling — allowing a patient with a blinding disease to know the risk for his or her children to develop the disease.

“You have got to first know the genes if you want to know how to manage the disease through manipulation of the gene or its consequence,” Dr. Lorenz said.

There are several known inheritance patterns: autosomal dominant, autosomal recessive and X-linked. It is difficult to identify the inheritance pattern of a specific retinal degeneration in an individual patient without family history, she said.

“Knowing the gene defect allows for genetic counselor to say, ‘You have an autosomal recessive disease, and if your spouse is not a carrier, your children will be all healthy.’ Knowing the gene or identifying the specific mutation in the patient allows you to give precise counsel as to inheritance and disease prognosis,” she said.

“It’s important to counsel the parents. You can be more specific if you know the mutation, for instance if it causes mild disease. You can tell the parents this is a stationary disease, for example. There is a visual problem, but your child will never be blind. Or, this is a disease where there will be progression, or a disease associated with mental retardation, because you can have combinations,” she said.

Ethical questions

As the possibility of a diagnosis based on genetic testing becomes more possible, ethical issues are also raised. One question is whether family members who do not exhibit the disease should be tested.

“In Germany, the Society of Human Genetics very clearly states that you should not do genetic testing in minors if there is no prospect for treatment,” she said.

This is particularly an issue for diseases such as Leber’s hereditary optic neuropathy, a mitochondrial disease. The disease has many more gene carriers than affected persons, but there is no means to know who will be affected.

Leber’s hereditary optic neuropathy is passed on only by women. All children of a woman who is a carrier will be carriers of the disease. But only about 10% of the daughters and 50% of the sons who are gene carriers will develop the disease.

The disease manifests as a relatively sudden onset of decreased vision in one eye followed by decreased vision in the second eye after a few days or months. Vision can be reduced to 20/200 or even to finger counting. There is no current therapy.

Once the clinical diagnosis has been made and molecular genetic testing confirms the disease, the clinician knows all siblings of this person and other people in the maternal line may carry the gene defect but may not develop the disease.

“How to handle this information is very difficult,” Dr. Lorenz said. “It’s a difficult situation for genetic counseling. You have identified the disease in a 20-year-old, and the family wants to know what the genetic situation is for the rest of the siblings. You can only cite the population statistics. You cannot say whether a given person will become diseased or not.”

Objective testing

Dr. Lorenz said that as more is learned and therapies move from experiment to clinical trials and then clinical practice, effective means to precisely describe the phenotype of a disease are also needed.

“If it is not a 100% cure, we will have to show that a specific therapy is able to slow the disease process. This is difficult because many measures can be conceived, but mainly are only subjective measures. Think about visual acuity or visual field. These are subjective tests. When you repeat the test you get variation. It’s difficult to tell whether there is improvement or whether there is just fluctuation,” she said.

Dr. Lorenz said objective tests used to monitor retinal disease also have limitations. For example, she said electroretinograms (ERGs) may be flat many years before the retina actually stops working.

“It’s just an electrical signal you can detect around a certain signal-to-noise ratio. The ERG, once it is no longer measurable, it is no longer useful for monitoring disease progression,” she said.

According to Dr. Lorenz, once the retina deteriorates to stages where ERGs are no longer useful, most means for monitoring retinal disease are subjective. This makes it difficult to be scientific because the data are variable and dependent on patient cooperation. Adequate personnel are also needed to perform the required examinations, which can be costly.

“Insurance will not pay for experimental protocols. This means a lot of research money in a laborious clinical investigation is needed. Identifying the gene is mainly lab work. Doing a good genotype to phenotype correlation means a lot of labor in the clinic,”
she said.

Early detection needed

According to sources, once a mutation and disease is fully described, any treatment would still require that the disease be detected early enough for the patient to maintain useful vision.

Dr. Lorenz noted that retinal diseases such as Leber’s congenital amaurosis are progressive diseases. Patients with specific forms of the disease can retain measurable visual acuity during childhood but become completely blind over time.

“The problem is that the most effective treatment would be in patients who still have measurable visual function. The patients with the most visual function would still be a minor age,” she said.

Dr. Lorenz explained that many so-called adult-onset hereditary retinal degenerations and diseases actually have their initial onsets during childhood. However, the symptoms of these diseases are so subtle and not functionally disabling during childhood that they are not recognized in children.

For example, Dr. Lorenz cited classical retinitis pigmentosa, which in most cases is genetic. She said if patients are asked carefully, most will have had some problems with night vision early in life.

The typical retinitis pigmentosa patient without a family history might notice the first symptoms at a stage at which the visual field is already constricted to 20°, she said.

“It’s so slow he or she doesn’t realize it. But when you ask, most people will tell you they had problems earlier. Parents noticed there were apparent visual field problems because the children did not react or would miss things on the floor, for example. If you listen carefully, you will notice that most of those hereditary retinal diseases have very early onset. I think it’s more a historical separation and that not all diseases are diagnosed in early childhood,” Dr. Lorenz said.

Current information on genetic retinal diseases can be found on the Internet on RetNet, most easily found by searching on the name.

For Your Information:

• John R. Heckenlively, MD, is Underwood Family Professor of Ophthalmology and director, Vision Genetics Center at the Jules Stein Eye Institute. He can be reached at 100 Stein Plaza, UCLA, Los Angeles CA 90095-7000 U.S.A.; +(1) 310-825-6089; e-mail: heckenlively@jsci.ucla.edu.
• Birgit Lorenz, MD, is a professor of ophthalmology and head of the department of pediatric ophthalmology, strabismology and ophthalmic genetics at the University of Regensburg. She can be reached at Franz-Josef-Strauss-Allee 11, 93042 Regensburg, Germany; +(49) 941-944-9220; fax: +(49) 941-944-9216; e-mail: birgit.lorenz@klinik.uni-regensburg.de.