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