DOHENY EYE INSTITUTE

Update for Brazil LHON Project with Special Emphasis on Brazil VII (2007)

Alfredo A. Sadun, MD, PhD

I was privileged to lead an international team of investigators to rural Brazil for a field investigation for the seventh time in October 5-9, 2007. This was a success, though in some ways particularly challenging. We focused on four main issues during the first three years of our project. 1) Genetics: We found and organized and defined the world’s largest pedigree of LHON established to be 11778, homoplasmic, J-haplogroup. 2) Epigenetics and epidemiology: We investigated whether there was an increased risk of converting from carrier to affected status with diet, toxins and exposure to smoking, drinking and non-tobacco smoke. 3) Clinical characteristics of this giant pedigree with LHON: We brought to rural Colatina several neuro-ophthalmologists and, using sophisticated equipment, performed comprehensive neuro-ophthalmological examinations. 4) Psychophysical testing: We developed and employed novel testing, including cutting-edge psychophysical evaluation techniques such as multifocal ERGs; the developers of this technology participated in this visit. We also investigated a number of new serological measures of oxidative stress.

Several conclusions were reached and published. These included the likelihood that both genetic and epigenetic factors effect conversion from carrier to affected status. Genetically, nuclear modifying genes influenced the penetrance rate of those affected. Environmental factors, including smoking and the consumption of alcohol more than doubled the risk of conversion.

We also concluded that there are subclinical manifestations of the disease that could be commonly described in carriers. Further, we could observe chronic progression in non-affected carriers. For example, we found alterations in mfERG, color vision and contrast sensitivity in psychophysical testing. We also noted retinal nerve fiber layer swelling that could be documented photographically or by GDx and that was reflected in subtle visual field changes. The disease is more chronic and complicated than commonly believed.

This led to our theory of pathophysiologic mechanisms. In short, the mutation of 11778 causes a defect of complex I that not only leads to a blockage of electron transport, but also to the production of reactive oxygen species (ROS). This changes the membrane potential of the mitochondria and induces an opening of the mitochondrial pores (MPTP) that liberates cytochrome C and this induces apoptotic death of the retinal ganglion cells.

Brazil IV-VI brought new revelations. We described first two and later four patients who were without visual complaint and therefore unaffected. They all demonstrated subclinical changes that showed progression until they led to conversion into an affected status of blindness. We also made arrangements for specialized necropsy of eye, brain and peripheral nerve tissues for some members of the family that may die during the next few years. We brought and added a new technology in the form of Optical Coherence Tomography (OCT) that offered the clinical quantification of retinal nerve fiber thickening and losses. Color vision and contrast sensitivity testing was done with Cambridge Colour Systems. Funduscopy was performed and comparisons made by two ophthalmologists with a third (me) for when there was a lack of complete agreement. Repeat ERGs, Visual Fields (Humphrey) and GDx were performed. We also repeated the OCT measures, and this was a great satisfaction as it was necessary to purchase this machine and transport it to/from Vitoria from Rio. These OCT studies were quite interesting in that they provided objective and quantifiable corroborations of the fundus examinations that some carriers were developing swelling of their retinal nerve fiber layers. Fundus photography rounded out the follow-up examinations.

As mentioned above, Brazil VII brought some new challenges including financial constraints. Support from International Foundation for Optic Nerve Diseases had to be slightly decreased due to the limits of fundraising. Unfortunately, this was largely aggravated by rising costs, mostly as a consequence of the falling U.S. dollar in comparison to the Brazilian reais, which reduced our purchasing power to less than half of what it was in 2002. Consequently, we had to scale back the number of investigators and some of the studies.

We decided to focus on three elements: 1) Further OCT studies. These would provide us clinically objective and reliable data on the nerve fiber layer swellings in LHON carriers and a means of watching these changes wax and wane over the years. 2) Blood testing for NSE. In the last two years we’ve found this to be a reliable index for those carriers most at risk for conversion to affected status and blindness. 3) Blood testing for axonal heavy chain neurofilament in the blood. In a paper now in preparation with Dr. John Guy of Gainesville, Florida, this has shown a dramatic elevation in LHON affected, and in carriers. Most remarkably, in carriers this correlates very closely with the many clinical and psychophysical changes we have been measuring. In short, we have found an excellent biomarker of this disease that can be used for any future clinical study of a therapeutic agent.

Finally, I should mention that all of these findings are leading us in the direction of a future clinical trial. We may have a partner in a small pharmaceutical company. They are looking into a new molecule that mimics the free radical scavenging properties of Idebenone, but that can be transported into mitochondria in much higher concentrations. This has tested well in cybrid studies performed in conjunction with Dr. Valerio Carelli in Italy, and they are now performing safety monitoring studies in rodents in England. If we are happy with the results, we can bring this agent to Brazil in 2008 and attempt a clinical trial using the quantitative tests described above as outcome measures.

Respectfully submitted,

Alfredo A. Sadun, MD, PhD

December 6, 2007

DOHENY EYE INSTITUTE

Update for Brazil LHON Project with Special Emphasis on Brazil VI (2006)

Alfredo A. Sadun, MD, PhD

For the sixth consecutive year, I was privileged to lead a large international team to rural Brazil for a field investigation. During the first three years we set out as three major goals to describe the genetics, epigenetics, epidemiology and clinical characteristics of this giant pedigree with LHON. In particular:

Brazil I - Find, organize and define the world's largest pedigree of LHON established to be 11778, homoplasmic, J-haplogroup. Furthermore, epidemiological studies and gene linkage analysis were initiated to determine the genetics and epigenetics of this pedigree.

Brazil II - Bring to rural (Colatina) Brazil, sophisticated equipment and world's experts in the use of this equipment for psychophysical examinations of members of this pedigree to complement the comprehensive neuro-ophthalmological examinations.

Brazil III - Develop novel testing including cutting edge psychophysical evaluation techniques such as multifocal ERGs and include the developers of this technology. We also investigated a number of new serological measures of oxidative stress.

At the end of these trips several conclusions had been reached and published. These included the fact that there were likely both genetic and epigenetic factors: Genetically, nuclear modifying genes influenced the penetrance rate of those affected. Environmental factors, including smoking and drinking (ETOH), also more than doubled the risk of conversion to affected status.

We also concluded that there were subclinical manifestations of the disease that allowed us to follow chronic progression in non-affected carriers. For example, we found alterations in mfERG, color vision, contrast sensitivity in psychophysical testing. We also noted retinal nerve fiber layer swelling that could be documented photographically or by GDx and that reflected itself in subtle visual field changes. The disease is more chronic and complicated than commonly believed. We also formulated a theory of pathophysiologic mechanisms. In short, the mutation of 11778 causes a defect of complex I that not only leads to a blockage of electron transport, but also to the production of reactive oxygen species (ROS). This changes the membrane potential of the mitochondria and induces an opening of the mitochondrial pores (MPTP) that liberates cytochrome C and this induces apoptotic death of the retinal ganglion cells.

Brazil IV also brought new revelations. In particular we found that we could follow the progression of LHON in carriers with these subclinical measures and that there were serological changes that were abnormal. Most remarkably, we described two patients, that were without visual complaint and therefore unaffected. These two not only demonstrated subclinical changes, but these showed progression as well as did serological tests (a rise in a specific blood test, neuron specific enolase--NSE). We also began treating one eye with brimonidine drops that were administered to the second eye before it became involved. These drops lead to increased levels of BCL-2 that should, theoretically, block the same MPTP pores in the mitochondria which we believe mediate the apoptotic death of the retinal ganglion cells that is the basis of the visual loss. Hence, although we haven't yet found a cure or even an effective therapy for the visual loss in LHON, we have found clinical measures that help predict who will lose vision and, furthermore, confirmation through a proof of principle, that our proposed mechanism of pathology, is likely to be correct.

Brazil V in 2005 allowed us to maintain the infrastructure of clinics and local volunteers who are available to examine the members of this pedigree year round and to respond to any carriers who develop visual loss. We also made arrangements for specialized necropsy of eye, brain and peripheral nerve tissues for some members of the family that may die during the next few years.

For the first time, we were able to apply new technology in the form of Optical Coherence Tomography (OCT) in the quantification of retinal nerve fiber thickening and losses. This was particularly useful to help us continue the longitudinal study of carriers both to document conversion to affected status and to see which parameters may predict such conversion as well as which parameters change with changes in epigenetic factors.

Brazil VI (October, 2006):

Constraints of resources, transportation and housing in Colatina required that we limit the number of investigators this year. Indeed, Drs. Sutter, Eells, Sherman and Roth were sensitive and kind enough to have postponed their projects to next year. At the last second, we did add one new investigator, however, given both a great new scientific opportunity and the NIH funding for pursuing this through Dr. Gino Cortopassi. Dr. Cortopassi had his PhD student, Jillian, come to draw bloods mostly to establish a library for mRNA obtained from the bloods of our pedigree. As an addition to our yearly studies, this gives us the opportunity to compare the genetic expression in carriers with those of affected patients to identify possible modifying or compensatory factors. For example, we think that there may be upregulation of those proteins needed to make myelin in carriers in an effort to reverse some of the changes of the disease. So, now we're looking at it differently. Instead of comparing risk factors (smoking, drinking, etc.), we are comparing inherent abilities to compensate as factors determining prevalence.

The other set of blood studies are complementary in two senses. Firstly, in that we will be analyzing the sera having separated off the cells for the project above. Secondly, we will be looking at neuron specific enolase (NSE) as a measure of neuronal distress. This will not only help as a marker of the disease to predict who is about to go blind, but as an outcome measure for future attempts in therapies.

We performed further comprehensive neuro-ophthalmological examinations in all the carriers and affected patients. These totaled 112, to which we added a few off-pedigree normal controls, but these were very few on the present field investigation. Color vision and contrast sensitivity testings were done with Cambridge Colour Systems. Funduscopy was performed and comparisons made by two ophthalmologists with a third (me) for when there was a lack of complete agreement. Repeat ERGs, Visual Fields (Humphrey) and GDx were performed.

We also repeated the OCT measures, and this was a great satisfaction as it was necessary to purchase this machine and transport it to/from Vitoria from Rio. These OCT studies were quite interesting in that they provided objective and quantifiable corroborations of the fundus examinations that some carriers were developing swelling of their retinal nerve fiber layers. Fundus photography rounded out the followup examinations. The OCT (anatomy), color vision (physiology) and NSE (biochemistry) may prove the most useful outcome measures against which to ascertain new treatment modalities.

After almost a week in Vitoria, the team took a bus to Vitoria. Here, like last year, we assembled in a conference room for two and a half days. Each group made brief powerpoint presentations of their data. Then, via multiple laptops with hookups to a central computer, the data was assembled and compared. Several groups began with analysis and the writing of manuscripts for peer review journals. To date, about nine publications document the new insights and discoveries made by our four previous field investigations.

One of the general conclusions drawn was that new measures of psychophysical changes could document different stages in the carrier with 11778 LHON. Accordingly, we are working on an algorithm that will generate a common scale to quantitate the extent of these subclinical changes in the carrier. These and about 7 other studies will be written up by December 3 to submit to this year's international ARVO meeting in the Spring.

Respectfully submitted,

Alfredo A. Sadun, MD, PhD

October 22, 2006

Summary of LHON Field Investigations made possible by IFOND support:

Brazil I- Brazil V

Alfredo A. Sadun, MD, PhD; July, 2006

In 2001, I was privileged to lead a large international team to rural Brazil for the first of five yearly field investigations for the understanding and cure of Leber's Hereditary Optic Neuropathy (LHON). LHON is tragic in that nothing can be currently done to prevent this catastrophic loss of vision that destroys the two optic nerves of young adults in the prime of their lives.

A great breakthrough occurred when, in the summer of 2001, we identified the world's largest pedigree living in a rural area of Brazil. This family contains about 332 subjects who are either affected with, carriers of or members of the nuclear family with LHON. Each year a large number of international and Brazilian specialists gather for very sophisticated testing and studies of these patients. This very intense period of investigation then leads to a number of new understandings that are presented and published in the world's literature.

The following is a simple summary of each of these yearly investigations, their objectives and accomplishments.

Brazil I - 2001: Epidemiological studies and gene linkage analysis were initiated to determine the genetics and epigenetics of this pedigree.

Brazil II - 2002: We brought to Brazil, sophisticated equipment and world experts in the use of this equipment for psychophysical examinations of members of this pedigree to complement the comprehensive neuro-ophthalmological examinations.

Brazil III - 2003: We developed novel testing including cutting edge psychophysical evaluation techniques such as multifocal ERGs. We also investigated a number of new serological measures of oxidative stress.

At the end of these trips several conclusions had been reached and published. We concluded that there were subclinical manifestations of the disease that allowed us to follow chronic progression in non-affected carriers. For example, we found alterations in color vision, contrast sensitivity and noted retinal nerve fiber layer swelling.

Brazil IV (2004) also brought new revelations. In particular we found that we could follow the progression of LHON in carriers, and that there were serological changes that were abnormal. Most remarkably, two patients, that were without visual complaint showed progression of these subclinical changes and both patients lost vision in a fairly classical way. However, these patients also demonstrated a delay before involvement of the second eye and a milder loss of vision in the second eye that may have reflected the effects of brimonidine drops that were administered to the second eye before it became involved. Hence, although we haven't yet found a cure or even an effective therapy for the visual loss in LHON, we have found clinical measures that help predict who will lose vision and, furthermore, confirmation through a proof of principle, that our proposed mechanism of pathology, is likely to be correct.

Brazil V (2005) brought an even larger number of scientists and clinicians. This trip pursued these objectives:

1. Maintain the infrastructure of clinics and local volunteers who are available to examine the members of this pedigree year round and to respond to any carriers who develop visual loss.

2. Arrange for specialized necropsy of eye, brain and peripheral nerve tissues for some members of the family that may die during the next few years.

3. Further refine the validity and reliability of several quantitative psychophysical tests.

4. Apply new technology in the form of Optical Coherence Tomography.

5. Obtain serum levels of NSE in a larger cohort of the pedigree.

6. Further apply the most sensitive new electophysiological measures.

7. Attempt new modalities of therapy such as long wavelength (670um) light to modulate mitochondrial activity in carriers.

8. Continue the longitudinal study of carriers both to document conversion to affected status and to see which parameters may predict such conversion.

9. Compare the results of various parameters with each other.

10. Educate and support members of the pedigree.

To date, about ten publications document the new insights and discoveries made by our five field investigations to rural Brazil to study LHON. Textbooks and book chapters are just now changing the description of LHON in light of these new studies. Great progress has been made, understandings established such that we now understand the basic biological cause of this disease.

About 15 international and renown scientists are joined by another 20 Brazilian volunteer scientists and physicians in this enterprise. They gladly donate several weeks each year for the privilege of working at the cutting edge of the science and hoping to soon bring about the successful management of this blinding disorder. All of these trips were largely subsidized by IFOND. We, the investigators, the study patients and all other patients around the world with LHON, and, most particularly the next generation who will inherit LHON but not go blind, are most grateful.

We thank the many supporters of IFOND who have made these groundbreaking investigations possible.

2)

The International Foundation for Optic Nerve Disease
P. O. Box 777, Cornwall NY 12518, USA.
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Email: ifond@aol.com
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