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