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IFOND Grant Proposal
June 5, 2017

Validation of objective outcome measures in LHON


 ^ Several clinical and psychophysical measures have been used to follow patients with Leber’s Hereditary Optic Neuropathy (LHON). Most of these measures are subjective and require patient input. In this study we propose the use of objective measures of electrical activity (PhNR) in vivo as markers of cellular function in LHON and experiments. We hope to establish an ideal protocol of outcome measures against which to test the effects of any purported treatment.

 ^  Electrophysiology


 ^   Leber’s Hereditary Optic Neuropathy (LHON) is a maternally inherited disease affecting Complex I of the respiratory supply chain (Carelli et al., 2004). The disease typically afflicts males in the second or third decade of life yet the underlying mitochondrial DNA (mtDNA) mutation is present from birth (Brown et al., 1992a, Brown et al., 1992b, Brown et al., 1992c). This incomplete penetrance is a peculiarity of LHON and illustrates that the mtDNA mutation is necessary but not sufficient to cause the optic neuropathy (Carelli et al., 2003). Studies have demonstrated that environmental factors such as smoking coupled with background mtDNA may influence disease penetrance (Carelli et al., 2006, Kirkman et al., 2009). However, it is still unclear who will become affected by the mutation and who will remain a carrier. Adding to this uncertainty is the variable expression of the disease in the affected population. Despite possessing the same mtDNA mutation some siblings appear to recover from the disease while others show no recovery (Chicani et al., 2013). This variability in clinical improvement presents a significant challenge when evaluating therapeutic interventions. Determining which patients are likely to respond would provide a clean homogenous patient population for future clinical trials.

PhNR as an Objective Marker for LHON.

 ^   The optic neuropathy present in LHON is the result of disruption to the retinal ganglion cell (RGC) layer. This disruption causes death of the axons and resulting in loss of retinal nerve fiber layers (RNFL). This results in a disconnect between the retina and the brain. Evaluation of RGC function is thus key in understanding the progression of the disease and the evaluating the effects of therapeutic interventions.

 ^   Currently ganglion cell function is predominantly evaluated using a number of subjective measures. Visual acuity, color vision and visual fields all rely on patient input and effort and are subjective. The advent of Optical Coherence Tomography (OCT) RNFL measurements have provided an objective measure of the axons involved in LHON. However the natural history of the disease creates a challenge for the use of RNFL measurements. The onset of conversion is marked by several finding including swelling of the RNFL. This swelling can persist for 6 months (Barboni et al., 2010) masking concurrent optic atrophy and as such OCT RNFL measurements become a poor biomarker for the initial phases of the disease onset. Part of this may be overcome by looking at the RGC layer itself (OCT GCC) which feeds the papillo-macula bundle which is the first area to show loss of RGC (Pan et al., 2012). The loss of ganglion cells and, by extension, the methods that are used to measure them are all markers of anatomical change, but they do not provide any insight into the level of disfunction present in these cells.

 ^   RGCs do produce a distinct electrical signal that can be isolated using electrophyiologic techniques. The most common of these techniques is to use pattern electroretinograms (pERG) and Holder showed that N95 component of the pERG is reduced in patients with LHON (Holder, 1997). The group at Bascom Palmer (Guy et al., 2014, Lam et al., 2014) recently demonstrated that pERG can be reliably recorded in patients with LHON. They note that PERG amplitude and phase were both low in the affected cohort and but could show any correlation in patients whose vision improved or worsened. One of the challenges with pERG is that patient need to fixate. This is an issue with patients who have LHON as they have lost central vision.

 ^   Another electrophysiological technique for assessing ganglion cell function was published by Viswanathan et al. (Viswanathan et al., 1999, Viswanathan et al., 2001). Viswanathan was able to identify a component of the flash ERG which specifically corresponded to RGC function. Called the photopic negative response (PhNR), it follows after the b wave. Experimentation with tetrodotoxin and a glaucoma model was able to show loss of this component of the ERG. Both these models demonstrate that the PhNR is the component of the flash ERG which reflects intact RGC activity.

 ^   Accurate objective quantification of RGC function would provide quantifiable metrics which could be useful to evaluate the effects of therapy. This is essential when attempting to quantify the disease severity not only in humans for the reason stated above, but also in animal models as the current subjective tests apply poorly to mouse models. In a comparison of PhNR and pERG in glaucoma patients, Preiser et al., (Preiser et al., 2013) found that both the PhNR and the pERG detected change in the RGC functions. But PhNR did have the advantage of not requiring clear optics or fixation which is a great benefit for animal testing and humans with central scotomas such as in LHON.

 ^   Thanks to funding from IFOND we have been able to follow patients in Brazil for over 15 years. During the most recent of these trips in 2015 we were able to obtain PhNR data from 17 carriers and 6 affected individuals from the Soave-Brazil pedigree. This data showed a significant difference between affected and carriers (Figure 1). The PhNR in control patients was also significantly larger than that of the affected and carrier population.

 ^   As a large well studied population the individuals of the Soave-Brazil pedigree are the ideal group of individuals to validate a new testing paradigm as potential confounders are controlled for and captured as part of the ongoing study. The purpose of this grant proposal is to return to Brazil to collect additional data and intertest data from members of the Soave-Brazil pedigree.


 ^  Hypothesis: the PhNR is consistently reduced in human LHON patients and reflects disease severity. We intend to test this hypothesis using the following experiments:

Quantification and qualification of the PhNR responses in patients with LHON.

A. To collect additional PhNR data from affected, carrier and control patients. The original study was limited due to the late arrival of the testing equipment. We were unable to test all willing members of the pedigree as a result. We therefore intend to collect data from the remaining members of the pedigree.

B. To determine the intertest variability of the PhNR in affected patients. As part of the validation of the technique it is important to understand what the intertest and intersession variability of the PhNR is in affected and carrier patients with LHON. To determine this, we will compare data collected in subjects with data collected from the first trip and repeat testing on multiple days in the same patient when possible.

 ^  Goal

These experiments will establish objective electrophysiological outcome measures for RGC function against which to later evaluate therapeutic measures in humans.

 ^  Budget and Timeline

 ^  This is intended to be a small team visit to Brazil. To help control costs and to allow for collection of data we are requesting funding for the team in Brazil and one international researcher to come to Brazil. The international researcher is Dr. Rustum Karanjia, the team from Brazil includes researchers from UNIFESP Adriana Berezovsky, Solange Salomao, Nivea Cavascan, Arthur Fernandes and Colatina including Dr. Milton Filho-Moraes and his team at the Colatina clinic. The trip is planned for the fall of 2017.


Barboni P, Carbonelli M, Savini G, Ramos Cdo V, Carta A, Berezovsky A, Salomao SR, Carelli V, Sadun AA (2010) Natural history of Leber's hereditary optic neuropathy: longitudinal analysis of the retinal nerve fiber layer by optical coherence tomography. Ophthalmology 117:623-627.

Brown MD, Voljavec AS, Lott MT, MacDonald I, Wallace DC (1992a) Leber's hereditary optic neuropathy: a model for mitochondrial neurodegenerative diseases. FASEB journal : official publication of the Federation of American Societies for Experimental Biology 6:2791-2799.

Brown MD, Voljavec AS, Lott MT, Torroni A, Yang CC, Wallace DC (1992b) Mitochondrial DNA complex I and III mutations associated with Leber's hereditary optic neuropathy. Genetics 130:163-173.

Brown MD, Yang CC, Trounce I, Torroni A, Lott MT, Wallace DC (1992c) A mitochondrial DNA variant, identified in Leber hereditary optic neuropathy patients, which extends the amino acid sequence of cytochrome c oxidase subunit I. American journal of human genetics 51:378-385.

Carelli V, Achilli A, Valentino ML, Rengo C, Semino O, Pala M, Olivieri A, Mattiazzi M, Pallotti F, Carrara F, Zeviani M, Leuzzi V, Carducci C, Valle G, Simionati B, Mendieta L, Salomao S, Belfort R, Jr., Sadun AA, Torroni A (2006) Haplogroup effects and recombination of mitochondrial DNA: novel clues from the analysis of Leber hereditary optic neuropathy pedigrees. American journal of human genetics 78:564-574.

Carelli V, Giordano C, d'Amati G (2003) Pathogenic expression of homoplasmic mtDNA mutations needs a complex nuclear-mitochondrial interaction. Trends in genetics : TIG 19:257-262.

Carelli V, Ross-Cisneros FN, Sadun AA (2004) Mitochondrial dysfunction as a cause of optic neuropathies. Progress in retinal and eye research 23:53-89.

Chicani CF, Chu ER, Miller G, Kelman SE, Sadun AA (2013) Comparing EPI-743 treatment in siblings with Leber's hereditary optic neuropathy mt14484 mutation. Canadian journal of ophthalmology Journal canadien d'ophtalmologie 48:e130-133.

Guy J, Feuer WJ, Porciatti V, Schiffman J, Abukhalil F, Vandenbroucke R, Rosa PR, Lam BL (2014) Retinal ganglion cell dysfunction in asymptomatic G11778A: Leber hereditary optic neuropathy. Investigative ophthalmology & visual science 55:841-848.

Holder GE (1997) The pattern electroretinogram in anterior visual pathway dysfunction and its relationship to the pattern visual evoked potential: a personal clinical review of 743 eyes. Eye 11 ( Pt 6):924-934.

Kirkman MA, Yu-Wai-Man P, Korsten A, Leonhardt M, Dimitriadis K, De Coo IF, Klopstock T, Chinnery PF (2009) Gene-environment interactions in Leber hereditary optic neuropathy. Brain : a journal of neurology 132:2317-2326.

Lam BL, Feuer WJ, Schiffman JC, Porciatti V, Vandenbroucke R, Rosa PR, Gregori G, Guy J (2014) Trial end points and natural history in patients with G11778A Leber hereditary optic neuropathy : preparation for gene therapy clinical trial. JAMA ophthalmology 132:428-436.

Lin CS, Sharpley MS, Fan W, Waymire KG, Sadun AA, Carelli V, Ross-Cisneros FN, Baciu P, Sung E, McManus MJ, Pan BX, Gil DW, Macgregor GR, Wallace DC (2012) Mouse mtDNA mutant model of Leber hereditary optic neuropathy. Proceedings of the National Academy of Sciences of the United States of America 109:20065-20070.

Pan BX, Ross-Cisneros FN, Carelli V, Rue KS, Salomao SR, Moraes-Filho MN, Moraes MN, Berezovsky A, Belfort R, Jr., Sadun AA (2012) Mathematically modeling the involvement of axons in Leber's hereditary optic neuropathy. Investigative ophthalmology & visual science 53:7608-7617.

Preiser D, Lagreze WA, Bach M, Poloschek CM (2013) Photopic negative response versus pattern electroretinogram in early glaucoma. Investigative ophthalmology & visual science 54:1182- 1191.

Viswanathan S, Frishman LJ, Robson JG, Harwerth RS, Smith EL, 3rd (1999) The photopic negative response of the macaque electroretinogram: reduction by experimental glaucoma. Investigative ophthalmology & visual science 40:1124-1136.

Viswanathan S, Frishman LJ, Robson JG, Walters JW (2001) The photopic negative response of the flash electroretinogram in primary open angle glaucoma. Investigative ophthalmology & visual science 42:514-522.

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