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Genetics and
Eye Disease
Though real
clinical impact remains a distant goal, these are exciting times for
genetics-based research into ocular disease.
Elias I. Traboulsi,
MD
Cleveland
This past summer, the world’s news media heralded a
scientific development that some have called more significant than the 1969
lunar landing. The completion of the first phase of the Human Genome Project
places medicine, and all of human society, at the threshold of a new era in the
diagnosis, treatment and prevention of major diseases. To be sure, many years
of research and significant obstacles lie ahead. This article, a continuation
of the series, “Ophthalmology’s New Century,” highlights some of
the research that’s going on today, and where some those efforts may lead
in the coming decades.
Monogenic Eye Diseases
Evidence that many common and rare diseases are genetically
determined has emerged over the last two centuries through the clinical
description of families, and the mapping of genes to specific chromosomal loci
and the identification of their structure and function. The elucidation of
biochemical and molecular pathways of embryological development and cellular
functions has paralleled these discoveries.
Two major groups of genetic eye
diseases can be identified. In the first or monogenic group, a specific gene
defect leads to the interruption of a biochemical or developmental pathway and
to the dysfunction of a group of cells that affects a predominant function.
A number of retinal
dystrophies, such as retinitis pigmentosa, Stargardt disease and Leber
congenital amaurosis, fall into this category. The genes that are involved in
the retinal dystrophies are predominantly or uniquely expressed in the retina,
and their mutations cause photoreceptor dysfunction and sometimes, even death.
Furthermore,
adjacent tissues such as retinal neurons, retinal pigment epithelium and
choroid become secondarily affected, and changes in these layers cause the
characteristic ophthalmoscopic signs of the individual disorder. Ocular
malformations such as aniridia and anterior segment dysgenesis can also be
classified into this group of monogenic disorders. They result from mutations
in transcription factors that determine the fate of embryonal cells and the
orderly differentiation of fetal structures. Other monogenic ocular diseases
include all the corneal dystrophies and the inherited types of infantile
cataracts.
Multifactorial Diseases
In
the second group of genetic eye diseases, multiple genes or a combination of
genes and environmental factors are involved. This group comprises some of the
more common conditions encountered in the clinical practice of ophthalmology,
such as age-related macular degeneration and adult-onset open-angle glaucoma. A
single gene may cause a significant predisposition for the disease, while other
genes or environmental factors lead to the development of disease
manifestations.
For
example, mutations in the gene for myocilin, a cause of juvenile glaucoma, are
more common in patients with adult-onset open angle glaucoma, and mutations in
the ABCR gene, a gene for Stargardt disease, are more common in patients with
age-related macular degeneration. Some mutation-carrying family members of
patients with these diseases do not develop the disease, lending support to the
presence of modifying genes or epigenetic factors in these individuals. The
presymptomatic detection of mutation carriers in such diseases will allow the
institution of rigid screening protocols and early therapy.
What’s Ahead
Technological advances such as the use of
robots, powerful molecular biology techniques and supercomputers, have allowed
world scientists to sequence more than 90 percent of the 100,000 or so human
genes through a massive effort sponsored by the National Institutes of Health,
the U.S. Department of Energy and private industry.
The generated data has allowed
scientists to move at a much faster pace in the matching of diseases and genes,
and to direct future efforts toward the identification of cellular and systemic
effects of gene mutations and the means of early detection and modification of
such effects.
Assays of micro-molecular biological reactions for the detection
of mutations or gene products will allow quick screening of patients for
abnormalities in panels of genes that have been linked to the genesis of their
disease. So-called micro-array or DNA chips will undoubtedly be in wide use in
the next few decades. Physicians will be able to order such tests on patients
with, among other diseases, cataracts, glaucoma, macular degeneration and
retinal dystrophies.
Doctors will be able to choose modalities of therapy based on this
information. Such therapy could include medications that are more effective in
certain disease genetic subtypes, micronutrient supplements that are effective
in some but not in other diseases, or the direct replacement of the gene
product using local or systemic administration.
A theoretical possibility is the use
of early surgical intervention in patients with types of glaucoma that are
known to be resistant to other forms of medical therapy. A discussion of the
field of gene therapy is beyond the scope of this article, but this modality of
treatment is a reasonable expectation in the 21st century. Another application
for rapid sequencing technology will be the detection of mutations in large
genes, such as the retinoblastoma gene, allowing precise prognostication for
the risk of developing additional retinal and systemic tumors.
The biotechnology industry has and
will continue to play a major role in the development of effective and
well-tolerated medications that will target specific biochemical and genetic
pathways. Some of these medications would selectively and specifically treat
disease subtypes. This may alleviate some of the uncertainties in therapeutic
responses that doctors and patients experience today, with diseases still being
lumped into large groups with common clinical findings but different
pathophysiologic and genetic mechanisms.
The great cost that will inevitably
be incurred to develop such therapies will hopefully be justified in the long
run by the availability of safe, efficacious and cost-effective medications
that will reduce the burden of blindness on society and enhance work
productivity.
It
will be possible in the not too distant future that a newly diagnosed patient
with a common disease such as glaucoma or macular degeneration would have a
blood sample drawn in the office of his/her ophthalmologist, DNA extracted,
serum proteins isolated, and a battery of tests for DNA or protein markers for
the various types of the disease rapidly done using sophisticated technologies.
The results of the tests would then be made available to the physician who
would prescribe the most appropriate therapy.
Dr. Traboulsi is the
head of Department of Pediatric Ophthalmology and director of the Center for
Genetic Eye Diseases at Cole Eye Institute, Cleveland Clinic.
Proteomic Approaches to Eye Disease
John W
Crabb, PhD
Cleveland
Despite our
growing knowledge of the human genome and of inherited visual disorders, the
molecular events underlying many eye diseases remain poorly understood. An
emerging technology that holds high promise for elucidating such molecular
events is proteomics.
What is proteomics? The proteome refers to the proteins expressed
by a genome in a cell type or tissue at a given point in time. The proteome is
much more complex than the genome, due to the different ways a gene may be
spliced during transcription and the numerous post-translational modifications
gene products undergo.
A few examples of such modifications include phosphorylation,
glycosylation, proteolytic processing, deamidation, sulfation and nitration.
Changes with age, the environment and state of health contribute further
complexity to the proteome.
Proteomics, then, is the global
analysis of gene expression at the protein level. Proteomic analyses pursue the
identification, quantification and characterization of expressed proteins. The
first step is the isolation of the cells or tissue of interest in order to
compare diseased vs. normal, young vs. old, drug treatment vs. control, etc.
For vision research, proteomic studies presently involve the collection of
donor eye tissues such as cornea, retina and retinal pigment epithelium. As we
learn more about systemic markers of eye disease, future proteomic screening of
plasma, urine or spinal fluid holds promise for diagnosing visual disorders.
Advances in
ophthalmic surgery may also lead to proteomic analyses of ocular biopsy
tissues. Sample preparation is a key parameter and directly influences the next
step in proteomic analyses, namely high resolution separation of the complex
mixture of proteins in the sample.
Current proteomic technology relies
heavily upon two-dimensional gel electrophoresis (isoelectric focusing followed
by SDS-polyacrylamide gel electrophoresis) for resolving protein mixtures.
Following 2-D gel separation, proteins are detected with various stains, and
gel spots are excised and treated with a protease (typically trypsin). The
resulting peptides are analyzed by mass spectrometry, yielding amino acid
sequence and/or peptide mass data; this provides the identity of the protein
via database searches.
A range of scanning devices and specialized software allow
qualitative and quantitative comparison among numerous 2-D gel images and for
databases of protein expression for a particular cell type or tissue to be
constructed .
Due
to the wide range of protein expression levels in cells (e.g., 10-106 copies
per cell) and variability in sample amount availability, researchers need other
separation methods besides 2-D gel technology to identify low-abundance
proteins. Today, ongoing methods development research continuously increases
the sensitivity, throughput and bioinformatic capability of proteome analysis.
Biotechnology projections claim that thousands of protein identifications per
hour will soon be possible.
How will proteomics benefit the
ophthalmologist? Of particularly urgent need in ophthalmology are more
effective therapies and methods for detecting a predisposition for
multifactorial diseases such as age-related macular degeneration (AMD). In such
diseases, environmental factors (for example, cigarette smoking, diet and
cholesterol level) as well as genetic factors contribute to the pathology.
Increased drusen content is a hallmark for the development of AMD, and current
proteomic approaches to identify drusen proteins may reveal new AMD drug
targets. Pharmaceutical companies are already using proteomics for global
analysis of protein expression in response to drug treatment (drug validation)
and drug discovery. Current proteomic screening of AMD and normal tissues also
has the potential to detect protein modifications (for example, specific types
of oxidative damage) that could be early warning markers for AMD.
In the future,
proteomic approaches to eye disease will facilitate the discovery of diagnostic
markers and drug targets and enhance opportunities for developing strategies to
predict then limit disease complications.
Dr. Crabb is a
researcher at the Cole Eye Institute, Cleveland Clinic.
|
An
International Perspective
Gearóid Tuohy, PhD
Dublin
The pace of molecular
biological research today is breathtaking. Each day brings more advanced
insights into previously mysterious maladies. Nowhere are these developments
more apparent than in the research programs in ocular disease.
The accelerated availability of
increasingly detailed maps and markers on the human genome has facilitated the
detection of both mutations in genes involved in ocular disease, and in
polymorphisms associated with a broad range of retinal dystrophies. These
databases are expanding exponentially, providing data that may potentially lead
to an increased number of therapeutic targets for interrupting the course of
these diseases. Advances in “high throughput” gene sequencing
technologies are already impinging on the etiologies of corneal dystrophies,
uveitis, glaucoma, refractive errors and retinal degenerations. All of these
possess very strong genetic components.
For example, The Ocular Genetics
Unit, Trinity College, Dublin, recently identified two novel gene mutations,
one responsible for juvenile open-angle glaucoma, and another responsible for
retinitis pigmentosa (RP). The earlier such conditions can be diagnosed, the
greater the number of options available to the patient, most importantly,
genetic counseling in the absence of a treatment.
Information technology will
similarly provide a wealth of information as researchers begin to compare
ocular-specific gene sequences found in a whole range of species. The
burgeoning field of bioinformatics is rapidly developing powerful tools to
extract meaningful information from vast quantities of data, leading to more
informed research activities. One example is the “RetNet” database of
mutations involved in retinal degenerative illnesses currently maintained by
Dr. Steve Daiger of the University of Houston. Such databases freely provide an
invaluable resource of information for researchers, clinicians and patients
(see www.sph.uth.tmc.edu/Retnet/sum-dis.htm).
The effort to develop new
genetically based therapeutic approaches to ocular diseases, and prevent
diseases before they develop, has produced some promising efforts and
collaborations internationally.
A recent research collaboration between Prof. Peter Humphries of
the Smurfit Institute of Genetics at Trinity College and Dr. Mario Capecchi of
the Howard Hughes Medical Institute at the University of Utah succeeded in the
development of a rhodopsin knockout mouse model for RP. This model has been
generated not only to provide a better understanding of the functional and
structural role of rhodopsin in the normal and diseased retina, but also to
provide a system in which the delivery of therapeutic genes may be tested. In
addition, these approaches strengthen the prospect of gene therapy as a viable
treatment for a variety of retinal dystrophies arising from either recessive
transmission or dominant haplo-insufficiency. Analysis of the Rho-/- knockout
demonstrated a severe degeneration of photoreceptor cells brought about through
a distinct form of cell death known as apoptosis. Apoptosis is an
energy-dependent program of cell death in which cells deconstruct themselves
silently without precipitating an immune reaction. Such a mode of cellular
degeneration has not only been demonstrated for retinal dystrophies but also
for a range of conditions such as Alzheimer’s, Huntington’s chorea
and many more. As a result, apoptosis is a booming segment of modern molecular
research.
Dr.
Herman Steller and associates at the Howard Hughes Medical Research Institute
at MIT, have been able to insert a specific gene into a fruit fly model of
human RP. The most significant finding to emerge from this research, aside from
preventing apoptotic cell death, was the demonstration of restored functional
vision to the retinas of these RP flies. In other words, it may be sufficient
to interrupt the apoptotic cell death in degenerating retinas to rescue disease
pathology without having to address the primary mutation.
A gene therapy originally designed to treat malignant tumors may
prove a valid therapeutic approach to posterior capsular opacification (PCO).
The technical problem that PCO presents is not dramatically different from what
oncologists face: Both conditions essentially address the problem of
restricting unwanted cellular growth.
At the Hôpital Puran, Toulouse
Cedex in France, Dr. Francois Malecaze and colleagues are focusing on the
thymidine kinase/ganciclovir system often referred to as “suicide gene
therapy.“ This therapy renders a transfected population of cells (in this
case the lens epithelial) susceptible to an otherwise nontoxic prodrug. In Dr.
Malecaze’s laboratory, researchers have achieved close to 100-percent
transfection efficiency in vivo. The study began by isolating the herpes
simplex virus type 1 thymidine kinase gene, excising it from the herpes simplex
virus and transferring it to the adeno-associated vector. Thymidine kinase is
generally active only in cells that are actively dividing and replicating their
DNA, such as lens epithelial cells on the surface of recently inserted
intraocular lenses. After transfecting to the lens epithelial cells of
experimental subjects, Dr. Malecaze’s team administered an intraocular
dose of the prodrug, ganciclovir. The thymidine kinase enzyme converts
ganciclovir to its monophosphate form, a conversion that does not normally
occur in eukaryotic cells. Consequently, DNA replication is halted and the
cells, no longer capable of dividing, begin to die. Dr. Malecaze has reported a
decrease in PCO in rabbits receiving this experimental therapy. The combination
of thymidine kinase activity and ganciclovir represents an effective mechanism
for distinguishing between target cells you wish to destroy and healthy cells
which, for the most part, remain unaffected. Although the adeno-associated
virus carrying thymidine kinase may gain access to cells other than the
actively dividing lens epithelial cells, the effect of the gene is confined to
cells attempting to replicate their DNA. Upon ganciclovir administration, cells
attempting to divide immediately mark themselves out for destruction. Dr.
Malecaze has been prompt to point out side effects, such as inflammation and
cytotoxicity. A number of technical difficulties must be addressed before this
particular gene therapy could progress to human trials. Yet, Dr.
Malecaze’s study clearly demonstrates a molecular-based approach targeting
the very fundamental biology of PCO. In the past five years, a significant
volume of research has evolved in the delivery of therapeutic genes to the eye
using viral vectors. Such therapeutic medicaments range from neurotrophic
growth factors and anti-tumor agents to ribozymes and recombinant tissue
plasminogen activators. In the field of ocular gene therapy, Dr. Matt La Vail,
at the University of California, San Francisco has shown that delivery of
ciliary neurotrophic growth factor to the retina of transgenic mice was able to
rescue degenerating photoreceptor cells. Similarly, Dr. Jean Bennett, at the
University of Pennsylvania’s Scheie Eye Institute, using replicative
deficient viruses, successfully delivered a number of other genetic constructs
to the mouse and primate retina, including one that blocks apoptosis. There is
good reason to believe that molecular genetic therapies are within our grasp.
While much of the research described here will not be appearing on a pharmacy
shelf tomorrow it is important to note, most especially for patients, that
these areas of research are both extremely active and relatively fast-moving.
The question is no longer ”if“ but “when?” Mr. Tuohy is in
the Department of Genetics, Trinity College, Dublin. |
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