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