Pupillometric analysis for assessment of gene therapy in Leber Congenital Amaurosis patients

Paolo Melillo, Leandro Pecchia, Francesco Testa, Settimio Rossi, Jean Bennett, Francesca Simonelli, Paolo Melillo, Leandro Pecchia, Francesco Testa, Settimio Rossi, Jean Bennett, Francesca Simonelli

Abstract

Background: Objective techniques to assess the amelioration of vision in patients with impaired visual function are needed to standardize efficacy assessment in gene therapy trials for ocular diseases. Pupillometry has been investigated in several diseases in order to provide objective information about the visual reflex pathway and has been adopted to quantify visual impairment in patients with Leber Congenital Amaurosis (LCA). In this paper, we describe detailed methods of pupillometric analysis and a case study on three Italian patients affected by Leber Congenital Amaurosis (LCA) involved in a gene therapy clinical trial at two follow-up time-points: 1 year and 3 years after therapy administration.

Methods: Pupillary light reflexes (PLR) were measured in patients who had received a unilateral subretinal injection in a clinical gene therapy trial. Pupil images were recorded simultaneously in both eyes with a commercial pupillometer and related software. A program was generated with MATLAB software in order to enable enhanced pupil detection with revision of the acquired images (correcting aberrations due to the inability of these severely visually impaired patients to fixate), and computation of the pupillometric parameters for each stimulus. Pupil detection was performed through Hough Transform and a non-parametric paired statistical test was adopted for comparison.

Results: The developed program provided correct pupil detection also for frames in which the pupil is not totally visible. Moreover, it provided an automatic computation of the pupillometric parameters for each stimulus and enabled semi-automatic revision of computerized detection, eliminating the need for the user to manually check frame by frame. With reference to the case study, the amplitude of pupillary constriction and the constriction velocity were increased in the right (treated eye) compared to the left (untreated) eye at both follow-up time-points, showing stability of the improved PLR in the treated eye.

Conclusions: Our method streamlined the pupillometric analyses and allowed rapid statistical analysis of a range of parameters associated with PLR. The results confirm that pupillometry is a useful objective measure for the assessment of therapeutic effect of gene therapy in patients with LCA.

Trial registration: ClinicalTrials.gov NCT00516477.

Figures

Figure 1
Figure 1
Description of the off-line pupil detection algorithm steps. (a) image acquired by the pupillometer; (b) image after the thresholding; (c) image after hole filling; (d) image after small object removing; (e) original image with the pupil border enhanced in white thanks to edge detection. (f) original image with the circle (in white) which fits the pupil border.
Figure 2
Figure 2
Pupil reflex of a subject involved in the study after a 0.2 second light stimulus triggered at zero second. The indicated parameters are computed as follows: BD, average of the pupil diameters 100 ms prior to the light stimulus (values to be averaged are in the green rectangular box): MA, the lowest pupil diameter after a light stimulus (represented by the orange point): RA, BD-MA (represented by the red segment); RT, tminimum amplitude- tstimulus (t = time) (represented by the light blue segment): CV, RA/RT (represented by the slope of the dark blue line).
Figure 3
Figure 3
Example of pupil diameter detection when the eyelashes are over the pupil (a) and on the subsequent frame in which the pupil is completely visible (b).

References

    1. Stone EM. Leber congenital amaurosis - a model for efficient genetic testing of heterogeneous disorders: LXIV Edward Jackson memorial lecture. Am J Ophthalmol. 2007;144:791–811. doi: 10.1016/j.ajo.2007.08.022.
    1. Banin E, Bandah-Rozenfeld D, Obolensky A, Cideciyan AV, Aleman TS, Marks-Ohana D, Sela M, Boye S, Sumaroka A, Roman AJ. et al.Molecular anthropology meets genetic medicine to treat blindness in the North African Jewish population: human gene therapy initiated in Israel. Hum Gene Ther. 2010;21:1749–1757. doi: 10.1089/hum.2010.047.
    1. Hauswirth WW, Aleman TS, Kaushal S, Cideciyan AV, Schwartz SB, Wang L, Conlon TJ, Boye SL, Flotte TR, Byrne BJ, Jacobson SG. Treatment of leber congenital amaurosis due to RPE65 mutations by ocular subretinal injection of adeno-associated virus gene vector: short-term results of a phase I trial. Hum Gene Ther. 2008;19:979–990. doi: 10.1089/hum.2008.107.
    1. Artur VC. Leber congenital amaurosis due to RPE65 mutations and its treatment with gene therapy. Prog Retin Eye Res. 2010;29:398–427. doi: 10.1016/j.preteyeres.2010.04.002.
    1. Bennett J, Ashtari M, Wellman J, Marshall KA, Cyckowski LL, Chung DC, McCague S, Pierce EA, Chen Y, Bennicelli JL. et al.AAV2 gene therapy readministration in three adults with congenital blindness. Sci Transl Med. 2012;4:120ra115.
    1. Maguire AM, High KA, Auricchio A, Wright JF, Pierce EA, Testa F, Mingozzi F, Bennicelli JL, Ying GS, Rossi S. et al.Age-dependent effects of RPE65 gene therapy for Leber’s congenital amaurosis: a phase 1 dose-escalation trial. Lancet. 2009;374:1597–1605. doi: 10.1016/S0140-6736(09)61836-5.
    1. Simonelli F, Maguire AM, Testa F, Pierce EA, Mingozzi F, Bennicelli JL, Rossi S, Marshall K, Banfi S, Surace EM. et al.Gene therapy for Leber’s congenital amaurosis is safe and effective through 1.5 years after vector administration. Mol Ther. 2010;18:643–650. doi: 10.1038/mt.2009.277.
    1. Maguire AM, Simonelli F, Pierce EA, Pugh EN Jr, Mingozzi F, Bennicelli J, Banfi S, Marshall KA, Testa F, Surace EM. et al.Safety and efficacy of gene transfer for Leber’s congenital amaurosis. N Engl J Med. 2008;358:2240–2248. doi: 10.1056/NEJMoa0802315.
    1. Fazzi E, Signorini SG, Scelsa B, Bova SM, Lanzi G. Leber’s congenital amaurosis: an update. Eur J Paediatr Neurol. 2003;7:13–22. doi: 10.1016/S1090-3798(02)00135-6.
    1. Aleman TS, Jacobson SG, Chico JD, Scott ML, Cheung AY, Windsor EA, Furushima M, Redmond TM, Bennett J, Palczewski K, Cideciyan AV. Impairment of the transient pupillary light reflex in Rpe65(-/-) mice and humans with leber congenital amaurosis. Invest Ophthalmol Vis Sci. 2004;45:1259–1271. doi: 10.1167/iovs.03-1230.
    1. Cideciyan AV, Hauswirth WW, Aleman TS, Kaushal S, Schwartz SB, Boye SL, Windsor EA, Conlon TJ, Sumaroka A, Pang JJ. et al.Human RPE65 gene therapy for Leber congenital amaurosis: persistence of early visual improvements and safety at 1 year. Hum Gene Ther. 2009;20:999–1004. doi: 10.1089/hum.2009.086.
    1. Bainbridge JW, Smith AJ, Barker SS, Robbie S, Henderson R, Balaggan K, Viswanathan A, Holder GE, Stockman A, Tyler N. et al.Effect of gene therapy on visual function in Leber’s congenital amaurosis. N Engl J Med. 2008;358:2231–2239. doi: 10.1056/NEJMoa0802268.
    1. Foster RG. Keeping an eye on the time: the Cogan lecture. Invest Ophthalmol Vis Sci. 2002;43:1286–1298.
    1. Aleman T, Scott M, Chen R, Chico J, Cheung A, Van Hooser J, Redmond T, Palczewski K, Jacobson S, Cideciyan A. Impairment of the pupillary light reflex in Rpe65-/- mice and patients with leber congenital amaurosis. Invest Ophthalmol Vis Sci. 2002;43:3490.
    1. Bergamin O, Zimmerman MB, Kardon RH. Pupil light reflex in normal and diseased eyes: diagnosis of visual dysfunction using waveform partitioning. Ophthalmology. 2003;110:106–114. doi: 10.1016/S0161-6420(02)01445-8.
    1. Kardon R, Kawasaki A, Miller NR. Origin of the relative afferent pupillary defect in optic tract lesions. Ophthalmology. 2006;113:1345–1353. doi: 10.1016/j.ophtha.2006.02.055.
    1. Volpe NJ, Dadvand L, Kim SK, Maguire MG, Ying G-S, Moster ML, Galetta SL. Computerized binocular pupillography of the swinging flashlight test detects afferent pupillary defects. Curr Eye Res. 2009;34:606–613. doi: 10.1080/02713680902993891.
    1. Soille P. Morphological image analysis: principles and applications. Berlin: Springer; 1999.
    1. Canver MC, Canver AC, Revere KE, Amado D, Bennett J, Chung DC. In: Bioengineering conference, proceedings of the 2010 IEEE 36th Annual Northeast; 26-28 March 2010. Samul K, editor. New York: IEEE; 2010. Algorithm for pupillometric data analysis; pp. 1–2.
    1. Kawasaki A, Moore P, Kardon RH. Variability of the relative afferent pupillary defect. Am J Ophthalmol. 1995;120:622–633.
    1. Dutsch M, Marthol H, Michelson G, Neundorfer B, Hilz MJ. Pupillography refines the diagnosis of diabetic autonomic neuropathy. J Neurol Sci. 2004;222:75–81. doi: 10.1016/j.jns.2004.04.008.
    1. Ferrari G, Marques J, Gandhi R, Heller S, Schneider F, Tesfaye S, Gamba H. Using dynamic pupillometry as a simple screening tool to detect autonomic neuropathy in patients with diabetes: a pilot study. Biomed Eng Online. 2010;9:26. doi: 10.1186/1475-925X-9-26.
    1. Smith SA, Dewhirst RR. A simple diagnostic test for pupillary abnormality in diabetic autonomic neuropathy. Diabet Med. 1986;3:38–41. doi: 10.1111/j.1464-5491.1986.tb00703.x.
    1. Smith SA, Smith SE. Reduced pupillary light reflexes in diabetic autonomic neuropathy. Diabetologia. 1983;24:330–332.
    1. Cideciyan AV, Aleman TS, Boye SL, Schwartz SB, Kaushal S, Roman AJ, Pang JJ, Sumaroka A, Windsor EA, Wilson JM. et al.Human gene therapy for RPE65 isomerase deficiency activates the retinoid cycle of vision but with slow rod kinetics. ProcNatlAcad Sci U S A. 2008;105:15112–15117. doi: 10.1073/pnas.0807027105.
    1. Cideciyan AV. Leber congenital amaurosis due to RPE65 mutations and its treatment with gene therapy. Prog Retin Eye Res. 2010;29:398–427. doi: 10.1016/j.preteyeres.2010.04.002.
    1. Kawasaki A, Munier FL, Leon L, Kardon RH. Pupillometric quantification of residual rod and cone activity in patients with visual loss due to Leber congenital amaurosis [abstract] Acta Ophthalmol. 2011;89:s248. doi: 10.1111/j.1755-3768.2009.01661.x.
    1. Zhu D, Moore ST, Raphan T. Robust pupil center detection using a curvature algorithm. Comput Methods Prog Biomed. 1999;59:145–157. doi: 10.1016/S0169-2607(98)00105-9.
    1. Iskander DR, Collins MJ, Mioschek S, Trunk M. Automatic pupillometry from digital images. IEEE Trans Biomed Eng. 2004;51:1619–1627. doi: 10.1109/TBME.2004.827546.
    1. Fotiou F, Fountoulakis KN, Goulas A, Alexopoulos L, Palikaras A. Automated standardized pupillometry with optical method for purposes of clinical practice and research. Clin Physiol. 2000;20:336–347. doi: 10.1046/j.1365-2281.2000.00259.x.
    1. Melillo P, Bracale M, Pecchia L. Nonlinear heart rate variability features for real-life stress detection. Case study: students under stress due to university examination. Biomed Eng Online. 2011;10:96. doi: 10.1186/1475-925X-10-96.
    1. Melillo P, Fusco R, Sansone M, Bracale M, Pecchia L. Discrimination power of long-term heart rate variability measures for chronic heart failure detection. Med Biol Eng Comput. 2011;49:67–74. doi: 10.1007/s11517-010-0728-5.
    1. Pecchia L, Melillo P, Sansone M, Bracale M. Discrimination power of short-term heart rate variability measures for CHF assessment. IEEE Trans Inf Technol Biomed. 2011;15:40–46.
    1. Pecchia L, Melillo P, Bracale M. Remote health monitoring of heart failure with data mining via CART method on HRV features. IEEE Trans Biomed Eng. 2011;58:800–804.

Source: PubMed

Szponzorok és közreműködők

Egészségi állapot

Kábítószer-beavatkozások

3
Iratkozz fel