- Email: [email protected]
Retinal Structure, Function, and Molecular Pathologic Features in Gyrate Atrophy
Retinal Structure, Function, and Molecular Pathologic Features in Gyrate Atrophy Panagiotis I. Sergouniotis, MD,1,2,* Alice E. Davidson, PhD,1,* Eva Lenassi, MD,1,2,* Sophie R. Devery, MSc,2 Anthony T. Moore, MA, FRCOphth,1,2 Andrew R. Webster, MD, FRCOphth1,2 Purpose: To describe phenotypic variability and to report novel mutational data in patients with gyrate atrophy. Design: Retrospective case series. Participants: Seven unrelated patients (10 to 52 years of age) with clinical and biochemical evidence of gyrate atrophy. Methods: Detailed ophthalmologic examination, fundus photography, fundus autofluorescence (FAF) imaging, spectral-domain optical coherence tomography, and microperimetry testing were performed. The coding region and intron– exon boundaries of ornithine aminotransferase (OAT) were analyzed. OAT mRNA was isolated from peripheral blood leucocytes of 1 patient and analyzed. Main Outcome Measures: OAT mutation status and resultant clinical, structural, and functional characteristics. Results: Funduscopy revealed circular areas of chorioretinal atrophy, and FAF imaging showed sharply demarcated areas of increased or preserved signal in all 7 patients. Spectral-domain optical coherence tomography revealed multiple intraretinal cystic spaces and hyperreflective deposit in the ganglion cell layer of all study subjects. Round tubular, rosette-like structures located in the outer nuclear layer of the retinae of the 4 older patients were observed (termed outer retinal tubulation). Thickening was evident in the foveolae of younger patients, despite the posterior pole appearing relatively preserved. Macular function, assessed by microperimetry, was preserved over areas of normal or increased autofluorescence. However, sensitivity was reduced even in structurally intact parts of the retina. The molecular pathologic features were determined in all study subjects: 9 mutations, 4 novel, were detected in the OAT gene. OAT mRNA was isolated from blood leukocytes, and monoallelic expression of a mutated allele was demonstrated in 1 patient. Conclusions: Fundus autofluorescence imaging can reveal the extent of neurosensory dysfunction in gyrate atrophy patients. Macular edema is a uniform finding; the fovea is relatively thick in early stages of disease and retinal tubulation is present in advanced disease. Analysis of leukocyte RNA complements the high sensitivity of conventional sequencing of genomic DNA for mutation detection in this gene. Financial Disclosure(s): The author(s) have no proprietary or commercial interest in any materials discussed in this article. Ophthalmology 2012;119:596 – 605 © 2012 by the American Academy of Ophthalmology.
Gyrate atrophy of the choroid and retina is a genetically determined, progressive condition associated with significantly increased plasma ornithine levels and inherited as an autosomal recessive trait.1 Patients typically report night blindness, loss of peripheral vision, or both in the second decade of life; myopia and early cataracts also are features of the disorder. On funduscopy, sharply demarcated, circular areas of chorioretinal atrophy distributed around the peripheral fundus are observed that, later in the disease process, coalesce and spread posteriorly. The macula and, consequently, central vision often is preserved into the 4th or 5th decade of life.2,3 More than 200 individuals with gyrate atrophy have been reported since it was first described in the late 19th century, with a significant proportion of them being Finnish.4 The underlying biochemical defect of gyrate atrophy is in ornithine aminotransferase (OAT), a vitamin B6-dependant mitochondrial matrix enzyme. Ornithine aminotransferase cat-
596
© 2012 by the American Academy of Ophthalmology Published by Elsevier Inc.
alyzes the conversion of L-ornithine, a nonproteinaceous amino acid, to proline and glutamic acid. Ornithine aminotransferase plays a role in cellular detoxification by disposing ornithine derived from dietary arginine, thus removing uncoupled ammonia.5– 8 Chronic reduction of plasma ornithine levels with an arginine-restricted diet has been reported to slow disease progression in some instances.9 The gene encoding the 439-amino acid human OAT precursor is expressed in most tissues, including liver, brain, neurosensory retina, and retinal pigment epithelium (RPE).10 More than 50 mutations have been reported in this gene (HGMD; http://www.hgmd.cf.ac.uk/; accessed June 2, 2011) with their deleterious consequences largely confined to the eye. Because the RPE is considered the primary site of dysfunction in gyrate atrophy, clinical trials of gene replacement therapy targeting the RPE or cellular therapeutic approaches involving induced pluripotent stem cell-derived RPE may be relevant to gyrate atrophy patients.11–14 ISSN 0161-6420/12/$–see front matter doi:10.1016/j.ophtha.2011.09.017
Sergouniotis et al 䡠 Retinal Structure and Function in Gyrate Atrophy Histopathologic study of postmortem tissue and in vivo cross-sectional imaging with time-domain (TD) optical coherence tomography (OCT) previously were used to provide insight into structural changes in gyrate atrophy.15–19 Retinal histopathologic analysis has been reported in human as well as in animal models of gyrate atrophy.11,15,20 In a 98-year-old patient, later identified to be homozygous for a p.Glu318Lys mutation,21 focal areas of photoreceptor atrophy with an underlying hyperplastic RPE were observed in the posterior pole. Near the transition from relatively preserved to atrophic retina, the RPE was either absent or dysmorphic. More peripherally to this transition zone, both RPE and photoreceptor cells were absent. At a short distance into the area of atrophic RPE, photoreceptor cell nuclei abutted directly onto Bruch’s membrane; the underlying choriocapillaris were preserved. Complete absence of outer retina, RPE, choriocapillaris, and most medium and large choroidal vessels was observed in the atrophic region.15 Regarding in vivo investigation, although resolution is relatively low in TD OCT detection, features such as edema and macular hole have been described.17–19 Spectraldomain (SD) OCT is an alternative to TD OCT with significant sensitivity and speed advantages, allowing highresolution imaging of tissue microstructure.22 Another noninvasive retinal imaging technology is fundus autofluorescence (FAF). Fundus autofluorescence uses naturally occurring fluorescence to provide an indicator of RPE structure and RPE and photoreceptor physiologic features.23,24 To assess the functional significance of abnormalities detected by SD OCT and FAF, testing of retinal function is required. Fundus-controlled microperimetry provides a method for accurate functional assessment of the central retina with high spatial resolution and continuous fixation monitoring.25 The present study describes the retinal structure and function in 7 patients with clinical, genetic, and biochemical evidence of gyrate atrophy. Novel FAF, SD OCT, and microperimetry findings are presented. Four novel mutations in the OAT gene are reported, and monoallelic expression of a mutated allele is demonstrated.
Patients and Methods Patients and Clinical Examination Seven unrelated patients with a diagnosis of gyrate atrophy were ascertained at Moorfields Eye Hospital, London, United Kingdom, over a 10-year period; all had a retinal dystrophy and funduscopic features compatible with those previously described in gyrate atrophy, together with significantly raised plasma ornithine levels. After informed consent was obtained, blood samples were collected and genomic DNA was extracted from peripheral blood leukocytes. The study was approved by the Moorfields and Whittington Hospitals local ethics committee, and all investigations were conducted in accordance with the principles of the Declaration of Helsinki. Patients 1 through 7 are numbered according to age, youngest to oldest. Clinical assessment included best-corrected visual acuity testing using the Early Treatment Diabetic Retinopathy Study charts, dilated fundus examination, color fundus photography (TRC-50IA; Topcon, Tokyo, Japan), FAF, SD OCT, and
fundus-controlled static microperimetry. The Spectralis HRA⫹OCT with viewing module version 5.1.2.0 (Heidelberg Engineering, Heidelberg, Germany) was used to acquire FAF images (over a 30⫻30° field, a 55⫻55° field, or both; 6 patients tested) and tomographs (all 7 patients tested). The SD OCT protocol included a horizontal, linear scan (100 B-scans averaged to improve the signal-to-noise ratio) centered on the fovea and a volume scan (minimum of 19 B-scan slices, 20⫻20° or 20⫻15°) for each eye. Retinal thickness in the central and inner macula Early Treatment Diabetic Retinopathy Study subfields were assessed; HEYEX software interface (version 1.6.2.0; Heidelberg Engineering) was used for all measurements. Outer retina morphologic feature interpretation was as suggested by Srinivasan et al.26 Static microperimetry (MP1 Microperimeter; Nidek Technologies, Padova, Italy) was performed in 6 patients. The semiautomatic examination with the number of stimuli ranging from 49 through 82, threshold sensitivities from 0 through 20 dB, test spot size Goldmann I, projection time of 1000 ms, and 4-2 staircase test strategy was used. A smaller than usual stimulus size was chosen because it previously was suggested that with such stimuli, it is possible to observe little islands with residual function.27
DNA Sequencing, mRNA Analysis, and Bioinformatics All 9 coding exons and flanking intronic boundaries of OAT were analyzed by direct sequencing of polymerase chain reaction (PCR) amplicons using the primers and conditions listed in Table 1 (available at http://aaojournal.org). When possible, parental DNA samples were screened to establish the phase of variants (patients 4 and 6). Leukocyte RNA was extracted from whole blood using a QIAamp RNA blood extraction kit (Qiagen, Crawley, United Kingdom) according to the manufacturer’s guidelines. cDNA was reverse-transcribed using a cDNA synthesis kit (BioLine, London, United Kingdom) with a random hexamer primer mix. For reversetranscriptase (RT) PCR reactions, OAT was amplified between exons 2 and 9 and between exons 6 and 10, respectively. The housekeeping gene -actin also was amplified as a positive control. Details of RT PCR primers used are listed in Table 2 (available at http://aaojournal.org). The identity of OAT RT PCR products was established by direct sequencing using standard procedures. Two different bioinformatics tools were applied to predict the effect of amino acid substitutions on OAT function: SIFT (version 4.0.3; available at http://sift.jcvi.org/; accessed June 2, 2011),28 which uses sequence homology to make predictions, and PolyPhen-2 (version 2.0.23; available at http://genetics.bwh.harvard.edu/pph2/; accessed June 2, 2011), which uses both sequence-based and structure-based predictive features.29
Results Clinical Findings Table 3 presents data on the 7 patients studied (age range, 10 –52 years). The mean visual acuity was 0.26 logarithm of the minimum angle of resolution units (range, 0.10 – 0.50 logarithm of the minimum angle of resolution units). Presentation was in the first decades of life: 4 patients were noted to have abnormal retinal appearance in routine refraction after difficulty reading the blackboard in early school years (patients 1, 2, 4, and 6); 1 patient had night vision problems at approximately 12 years of age (patient 5); 1 patient recalled night blindness from a young age and problems
597
Ophthalmology Volume 119, Number 3, March 2012 Table 3. Clinical Characteristics and Molecular Pathologic
Refractive Error (Diopters) Patient No.
Gender
Age at Examination (yrs)
1 2 3 4 5 6 7
F F F M F F F
10 14 17 30 34 38 52
Right Eye
Left Eye
⫺6.75/⫺4.00⫻180* ⫺6.50/⫺4.00⫻180* ⫺6.00/⫺2.50⫻25 ⫺5.50/⫺2.50⫻155 ⫺2.00 ⫺1.75/⫺0.50⫻170 Myopic astigmatism ⫺13.50/⫺1.50⫻75† ⫺11.00/⫺1.50⫻120† Myopic astigmatism† ⫺9.50/⫺2.00⫻177† ⫺7.75/⫺3.00⫻7†
Visual Acuity (Logarithm of the Minimum Angle of Resolution) Right Eye
Left Eye
0.34 0.10 0.22 0.18 0.50 0.10 0.50
0.30 0.16 0.20 0.20 0.20 0.40 0.20
F ⫽ female; HRR ⫽ Hardy-Rand-Rittler device; ISH ⫽ Ishihara color vision test; M ⫽ male. Color vision was evaluated using the HRR (American Optical Company, New York, NY) with both eyes open or the 24-plate ISH (Kanehara and Co. *Cycloplegic refraction. † Prior cataract surgery. ‡ Diagnosed with retinal dystrophy; gyrate atrophy was diagnosed at age 30 years.
with peripheral vision from early teenage years, but medical advice was sought only at age 26 years (patient 7); and 1 patient had poor central vision (1.00 logarithm of the minimum angle of resolution units in each eye) with electrophysiology, suggesting that this was the result of functional visual loss. Six of 7 patients were night blind, and 7 of 7 had elevated plasma ornithine concentration. Patients 5 and 7 underwent bilateral cataract extractions with intraocular lens implants at 29 and 41 years of age, respectively; patient 6 underwent surgery in the right eye operated at 22 years of age and in the left eye at 26 years of age. Patient 7 had late-onset sensorineural hearing loss and a paternal family history of hearing impairment; hearing problems also were reported for patient 1. All patients had a negative family history of retinal disease; patients 1 and 2 were born to consanguineous parents. Funduscopy revealed typical changes of gyrate atrophy in all 7 patients. Importantly, retinal pigment migration was minimal or absent and retinal vessel diameter was relatively preserved. Sharply demarcated areas of increased or preserved signal were observed in FAF images of all 6 cases tested. Color fundus photography and FAF imaging are presented in Figure 1. Spectral-domain OCT demonstrated structural changes that were concordant between eyes in all patients. For patients 1 and 3, SD OCT revealed multiple intraretinal cystic spaces bilaterally and no other significant abnormality (Fig 2). Hyporeflective cysts varying in size also were detected in the remaining 5 cases. In these patients (patients 2, 4, 5, 6, and 7), the hyperreflective band corresponding to the photoreceptor inner segment and outer segment (IS/OS) junction was preserved in the regions where autofluorescence signal was normal or increased. Outside of these regions, typically at the edges of foveal scans and at the more peripheral scans of the volume series, extensive loss of IS/OS junction reflectivity and significant thinning were observed. The thinning was not confined to the scattering layers in the outer retina, but also extended to the inner retinal layers, the hyperreflective band corresponding to the RPE–Bruch’s complex, and the structures often seen posterior to it. Structures corresponding to the external choroidal margins, large choroidal vessels, and inner scleral surface were visible on the scans of all patients. A loss of focal hyperreflectivity in the inner choroid was observed frequently, and structures resembling large choroidal vessels appeared to be in close proximity to the hyperreflective band interpreted as Bruch’s membrane. Vitreomacular traction, epiretinal membrane, or both, causing irregularities of the retinal surface, were observed in the 4 oldest patients. An interesting shared
598
feature in the SD OCT images of all 7 patients was the presence of hyperreflective deposit in the ganglion cell layer, particularly over the regions of preserved autofluorescence or at a short distance into the regions of absent autofluorescence. When the amount of hyperreflective material was increased, deposits coalesced and were depicted as vertical hyperreflective lines (for example Fig 2, patients 3 and 4; Fig 3B, patient 4, available at http://aaojournal.org). Another interesting finding was the presence of round or ovoid hyporeflective spaces with hyperreflective borders on SD OCT scans from the 4 older patients (patients 4, 5, 6, and 7). These typically were found in the transition from relatively preserved to atrophic retina (Fig 4A, patient 4) and previously were termed outer retinal tubulation.30 Finally, when thickness profiles were evaluated, relative thickening was observed in the more central Early Treatment Diabetic Retinopathy Study subfields of younger patients (Fig 3A, patients 1, 2, and 3, available at http://aaojournal.org). Static microperimetry testing results were overlaid with FAF and SD OCT and are presented in Figure 4. Stable fixation was observed in all eyes tested. In patients with structurally preserved posterior pole, although appropriate normative data are lacking, a mild reduction in macular sensitivity is likely (Fig 4A-B, patients 1 and 3). In the more severely affected individuals (patients 4 through 7), reduced sensitivity was evident over regions of increased or preserved autofluorescence signal (on FAF) and relatively intact IS/OS junction line (on SD OCT). In 6 of 8 eyes, outside these areas, patients did not respond even to the brightest stimuli (Fig 4A-B, patients 4, 5, 6, and 7). Interestingly, in the remaining 2 eyes, consistent responses were documented over areas with significant outer retinal loss (absent IS/OS junction line on SD OCT) and RPE loss (no signal on FAF; Fig 4C, patients 4 and 7).
Molecular Findings Overall, 9 different likely disease-causing DNA variants in OAT were detected in 13 alleles of 7 patients and included 7 missense and 2 nonsense changes (Table 3). The predicted effects of each of the 7 amino acid substitutions are summarized in Table 4.21,31,32 The bioinformatics programs SIFT and PolyPhen-2 both supported the likely pathogenicity of each of the identified missense mutations, including p.Gly51Asp, p.Gly91Glu, and p.Gly121Asp, which are novel to this study. Furthermore, all novel mutations identified (p.Gly51Asp, p.Gly91Glu, p.Gly121Asp, and p.Arg250X) were found to be absent from the
Sergouniotis et al 䡠 Retinal Structure and Function in Gyrate Atrophy Features of Patients with Gyrate Atrophy
Color Vision
Presenting Symptom (Age at Diagnosis [yrs])
Nyctalopia (Age First Noted [yrs])
Ornithine Levels at Diagnosis (mol/l)
Molecular Diagnosis, Mutations in OAT
HRR full HRR full ISH 17/17 ISH 17/17 ISH test plate only ISH 6/17 ISH 5/17
Increasing myopia (9) Increasing myopia (11) Poor central vision (17) Increasing myopia (6‡) Night blindness (13) Increasing myopia (9) Night blindness (26)
Yes (8) No Yes (17) Yes (14) Yes (12) Yes (27) Yes (always)
1390 1152 775 908 ⬃900 ⬃1000 ⬃700
p.[Gly91Glu];[Gly91Glu] p.[Gly51Asp];[Gly51Asp] p.[Glu318Lys];[?] p.[Pro199Gln];[Pro417Leu] p.[Pro199Gln(;)Pro417Leu] p.[Gly121Asp];[Tyr299X] p.[Tyr55His(;)Arg250X]
Ltd, Tokyo, Japan) with each eye; the best score is presented. Normal plasma ornithine levels: 20 to 114 mol/l. Novel changes are in boldface.
1000 genomes project data set (628 individuals from the 20100804 sequence and alignment release). Because patient 3 was identified as harboring only a single heterozygous missense mutation in OAT (p.Glu318Lys), patient leukocyte mRNA was analyzed by RT PCR. Two pairs of RT PCR primers were designed to amplify between OAT exons 2 through 9 and between exons 6 through 10, respectively (Table 2, available at http://aaojournal.org). The PCR products of the expected sizes (970 bp and 596 bp) were amplified for both control and patient samples (Fig 5A, available at http://aaojournal.org). Direct sequencing of the RT PCR products demonstrated the patient apparently to be homozygous for the likely pathogenic p.Glu318Lys variant, which previously was identified in the heterozygous state by direct sequencing of genomic DNA. In addition, RT PCR products revealed apparent homozygosity for a single nucleotide polymorphism (c.1134C¡T, p.(⫽); rs11461), which had been identified in the heterozygous state when sequencing the patient’s genomic DNA (Fig 5B, available at http://aaojournal.org). These data strongly suggest monoallelic expression of the disease-causing allele (p.Glu318Lys), which is likely to be the result of an unidentified sequence variant abrogating expression from one of the patient’s chromosomes (Fig 5C, available at http://aaojournal.org).
Discussion This report describes retinal structure and function in 7 gyrate atrophy patients and reports 4 novel mutations. Novel data related to imaging studies (FAF and SD OCT) are presented, providing insights into the anatomic changes at both early and late stages of gyrate atrophy. Microperimetry testing results are reported, and the relationship between structural abnormalities and functional deficits is evaluated. All 7 study patients had myopic refraction, fundus changes typical of gyrate atrophy, and greatly elevated plasma ornithine levels. Six of 7 patients reported poor night
vision, increasing myopia, or both as their first symptom(s). In a previous report, more than 70% of subjects presented with increasing myopia at presentation.4 Funduscopy through dilated pupils in highly myopic children with or without night blindness is key for early diagnosis of gyrate atrophy. However, it is noted that presentation can be unusual, as in patient 3, an individual with low myopia with poor central vision, probably unrelated to the primary retinopathy. Visual acuity loss in patients with gyrate atrophy can be the result of disease progression causing central photoreceptor cell death, but also cataract or macular edema. By the second decade, cataract is considered a uniform finding in gyrate atrophy patients.33 Cataracts typically are posterior sutural, may have unique histologic characteristics, and require surgical intervention by the third or fourth decade of life.3,33 In the present series, the 3 older patients (patients 5, 6, and 7) already had undergone bilateral cataract surgery. Cystoid macular edema can be another ocular manifestation of gyrate atrophy, with at least 4 cases reported to date; fluorescein angiography and TD OCT have been used to confirm this macular complication.17,18 Hyporeflective cystic spaces, predominantly at the inner retina, were identified with SD OCT in all patients, suggesting that edema also is a uniform finding. Retinal pigment epithelial dysfunction or loss leading to disruption of the outer blood–retinal barrier and diffusion of fluid toward intraretinal spaces is likely to be the main mechanism of edema formation in gyrate atrophy. The fact that it is observed even in younger patients with structurally intact, although thickened (Fig 3, available at http://aaojournal.org), retina may suggest RPE dysfunction as an early abnormality leading to photoreceptor cell exposure to toxic agents from choroidal circulation. In older patients, in addition to the inner retinal hyporeflective cystic
™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™3 Figure 1. Color fundus photography and fundus autofluorescence (FAF) imaging of patients with gyrate atrophy and OAT mutations. The number relating to each pair of images corresponds to the case number (patients 1 through 7) and patients are numbered according to age (youngest to oldest; age range, 10 –52 years). Bone spicule pigmentation is minimal, retinal vessels are relatively preserved, and circular areas of chorioretinal atrophy are evident in fundus photographs. Sharply demarcated areas of increased or normal signal are observed in FAF images.
599
Ophthalmology Volume 119, Number 3, March 2012
600
Figure 2. Foveal linear spectral-domain optical coherence tomography scans of 7 patients with gyrate atrophy (patients 1 through 7, ordered by age).
601
Ophthalmology Volume 119, Number 3, March 2012
602
Sergouniotis et al 䡠 Retinal Structure and Function in Gyrate Atrophy Table 4. Missense Coding Sequence Changes in OAT That Are Likely to be Pathogenic and In Silico Analysis of Their Effect SIFT
Polyphen-2
Nucleotide Substitution
Amino Acid Exchange
Prediction
Tolerance Index (0–1)
Prediction
HumVar Score (0–1)
Reference
c.152 G¡A c.163 T¡C c.272 G¡A c.362 G¡A c.596 C¡A c.952 G¡A c.1250 C¡T
p.Gly51Asp p.Tyr55His p.Gly91Glu p.Gly121Asp p.Pro199Gln p.Glu318Lys p.Pro417Leu
Intolerant Intolerant Intolerant Intolerant Intolerant Intolerant Intolerant
0.00 0.00 0.00 0.01 0.00 0.02 0.00
POS PRD PRD PRD PRD POS PRD
0.867 0.980 1.000 0.995 0.994 0.626 0.997
This study Kaufman et al., 199031 This study This study Kaufman et al., 199031 Mashima et al., 199921 Brody et al., 199232
POS ⫽ possibly damaging; PRD ⫽ probably damaging; SIFT ⫽ sorting intolerant from tolerant (http://sift.jcvi.org/). SIFT results are reported to be tolerant if the tolerance index is ⱖ0.05 or intolerant if tolerance index is ⬍0.05. Polyphen-2 (version 2.1.0) appraises mutations qualitatively as benign, POS, or PRD based on the model’s false-positive rate. The cDNA is numbered according to Ensembl transcript ID ENST00000368845, in which ⫹1 is the A of the translation start codon.
spaces, SD OCT detected epiretinal membrane and vitreomacular traction. Hyperreflective deposits on SD OCT were observed in the ganglion cell layer of all gyrate atrophy patients in this report (Fig 2, patients 3 and 4; Fig 3B, patient 4, available at http://aaojournal.org). This material was more obvious but not confined to the areas near the transition from relatively preserved to atrophic retina. Colorless, elongated, glittering crystals, better seen in the fluorescein angiogram and located over or proximal to the preserved areas, have been described previously in gyrate atrophy patients.2,34 Immunofluorescent localization of glial fibrillary acidic protein in the Oat–/– mouse retina revealed labeling of the astrocytes in the innermost retina.11 This finding suggests gliotic response to ongoing cell death,35 and one possibility is that the hyperreflective deposits on SD OCT reflect retinal gliosis. Inner retinal globular hyperreflective aggregates, with some similarities to those described in this study, previously were reported in eyes with acute zonal occult outer retinopathy.36 Another interesting finding in the tomographs of older gyrate atrophy patients was the presence of outer retinal tubulation (Fig 4, patient 4). Similar structures have been described previously in Bietti crystalline dystrophy, pseudoxanthoma elasticum, and age-related macular degeneration.30 It has been proposed that these rosettes are formed by degenerating photoreceptor cells becoming arranged in a
circular or ovoid fashion.30 This may explain the relative preservation of visual function over these structures (Fig 4, patient 4). Because RPE is an early site of dysfunction in all conditions mentioned above, the authors propose that the first step in the process may be RPE loss. In support of this is evidence from transplantation studies in murine eyes, which suggests that symbiosis of photoreceptors with microglia in rosettes promotes photoreceptor cell survival in the absence of RPE.37 This study showed that in gyrate atrophy patients, microperimetry stimuli are recognized over areas of preserved or increased signal in FAF. Surprisingly, in 2 eyes of 2 patients (Fig 4B), preserved retinal function also was observed over areas of absent autofluorescence signal, corresponding to RPE atrophy. When the relevant microperimetry data points were overlaid with SD OCT, a very thin retina with complete loss of the IS/OS junction line was seen (Fig 4C). It can be speculated that reflection, or scatter, of the stimulus from the sclera onto preserved and functioning anterior retina may account for the absence of an absolute scotoma, which would be expected from an area of complete loss of RPE and outer retina. Of the 2 eyes concerned, the results were reproducible and not evidently the result of poor compliance. When the patients were asked to report the direction of the stimuli, again this was reported reproducibly, but did not always map to the area stimulated. Each eye had areas of intact anterior retina, and the most
4™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™ Figure 4. Functional assessment of the central retina in 6 patients with gyrate atrophy. A, Static microperimetry testing results overlaid with fundus autofluorescence (FAF) images and spectral-domain optical coherence tomography (SD OCT) scans from patient 3 (17 years of age) and patient 4 (30 years of age) are presented. Mild reduction of foveal sensitivity is observed in the younger patient, for whom the FAF image shows normal results and the SD OCT scan shows only some cystic spaces. In the older patient, sensitivity is reduced over areas of relatively preserved FAF and with an intact inner segment– outer segment junction line. However, outside these regions, there is no response even to the brightest stimulus. The asterisk marks a round hyporeflective space with hyperreflective borders (retinal tubulation). This may reflect photoreceptors forming rosettes in the absence of retinal pigment epithelium. B, Static microperimetry results from patient 1 (10 years of age), patient 5 (34 years of age), patient 6 (38 years of age), and patient 7 (52 years of age). Data sets have been overlaid with infrared (patient 1) or FAF (patients 5, 6, and 7) images. Similarly to patient 3, patient 1 has mildly reduced retinal sensitivity despite structurally intact retina. Conversely, in the older patients, the retina is functional only over areas of preserved autofluorescence, thus highlighting the clinical value of FAF in gyrate atrophy. C, Static microperimetry testing results overlaid with FAF images and SD OCT scans from patient 4 (30 years of age) and patient 7 (52 years of age) are presented. Surprisingly, retinal function appears to be preserved over areas of absent autofluorescence signal (on FAF) and very thin outer and total retina (on SD OCT). The scleral surface is clearly visible, and the presence of islands of preserved photoreceptors cannot be excluded (arrowhead).
603
Ophthalmology Volume 119, Number 3, March 2012 likely explanation is that scatter toward the functioning areas allowed the patient to see these stimuli. Alternative explanations include small areas of preserved retina missed by OCT and FAF imaging, and inner-retinal phototransduction. This finding suggests the need for caution in the interpretation of microperimetry data when investigating dystrophies such as gyrate atrophy, and is particularly relevant to its use in characterizing the deficit in choroideremia in preparation for relevant therapeutic trials. Six of 7 patients in this study were identified as harboring either proven (patients 1, 2, 4, and 6) or presumed (patients 5 and 7) biallelic mutations in OAT. Patients 4 and 5, who are not knowingly related, share the same genotype. The observed phenotypic differences between the 2 patients most likely can be attributed to other currently unidentified genetic factors, environmental modifying factors, or both. Patient 3 was found to harbor only a single previously reported21 heterozygous OAT mutation, p.Glu318Lys. Interestingly, patient 3 is of British ancestry, and all previously reported individuals carrying this mutation also are of British or Irish descent.21 Because gyrate atrophy is a recessive condition caused by biallelic mutations, the patient’s leukocyte mRNA was analyzed by RT PCR to establish whether OAT allelic expression was abnormal. Although the correctly sized RT PCR products were amplified from the patient sample, direct sequencing of the amplimers demonstrated monoallelic expression of the disease-causing allele (Fig 4). The underlying cause of this loss of expression has not been identified; however, it may represent disruption of the OAT promoter, mutation of a regulatory element potentially distant from the coding sequence, or an undetected intronic mutation.38,39 This finding demonstrates that it is possible to amplify OAT mRNA from peripheral blood leucocytes, and thus to investigate potential alterations in pre-mRNA splicing, expression of OAT for diagnostic purposes, or both. In addition, this has important implications for future gyrate atrophy diagnostic studies because OAT allelic gene expression analysis has the potential to indicate the presence of mutations that will be missed by conventional mutation detection methods. The Glu318 residue is important for pyridoxal phosphate (the active form of vitamin B6) cofactor binding to OAT,7 and it has been suggested previously that a p.Glu318Lys substitution disrupts it.15,21 This missense change is one of only a handful of OAT mutations demonstrated to be responsive to pyridoxal phosphate supplementation.21,40 – 42 Importantly, gyrate atrophy patients reported with pyridoxal phosphate-responsive genotypes have been noted to have a less severe and progressive clinical presentation,21 a finding in keeping with the clinical presentation of patient 3 in this study. In conclusion, the present study expands our knowledge of gyrate atrophy. Structural changes are reported in detail: macular edema is a common finding, the fovea is relatively thick in early stages of disease, deposits that may suggest retinal gliosis are observed, and retinal tubulation is present in advanced disease. Functional testing highlights the importance of FAF as a monitoring tool for gyrate atrophy and suggests that retinal sensitivity can be reduced even over structurally intact retina and in early stages of disease pro-
604
gression. Allelic heterogeneity is high in the disorder, and leukocyte RNA analysis can increase the sensitivity of mutation detection.
References 1. Simell O, Takki K. Raised plasma-ornithine and gyrate atrophy of the choroid and retina. Lancet 1973;1:1031–3. 2. Takki K. Gyrate atrophy of the choroid and retina associated with hyperornithinaemia. Br J Ophthalmol 1974;58:3–23. 3. Takki KK, Milton RC. The natural history of gyrate atrophy of the choroid and retina. Ophthalmology 1981;88:292–301. 4. Peltola KE, Nanto-Salonen K, Heinonen OJ, et al. Ophthalmologic heterogeneity in subjects with gyrate atrophy of choroid and retina harboring the L402P mutation of ornithine aminotransferase. Ophthalmology 2001;108:721–9. 5. O’Donnell JJ, Sandman RP, Martin SR. Gyrate atrophy of the retina: inborn error of L-ornithin:2-oxoacid aminotransferase. Science 1978;200:200 –1. 6. Volpe P, Sawamura R, Strecker HJ. Control of ornithin deltatransaminase in rat liver and kidney. J Biol Chem 1969;244: 719 –26. 7. Shen BW, Hennig M, Hohenester E, et al. Crystal structure of human recombinant ornithine aminotransferase. J Mol Biol 1998;277:81–102. 8. Valle D, Walser M, Brusilow SW, Kaiser-Kupfer M. Gyrate atrophy of the choroid and retina: amino acid metabolism and correction of hyperornithinemia with an arginine-deficient diet. J Clin Invest 1980;65:371– 8. 9. Kaiser-Kupfer MI, Caruso RC, Valle D. Gyrate atrophy of the choroid and retina: further experience with long-term reduction of ornithine levels in children. Arch Ophthalmol 2002; 120:146 –53. 10. Rao GN, Cotlier E. Ornithine delta-aminotransferase activity in retina and other tissues. Neurochem Res 1984;9:555– 62. 11. Wang T, Milam AH, Steel G, Valle D. A mouse model of gyrate atrophy of the choroid and retina: early retinal pigment epithelium damage and progressive retinal degeneration. J Clin Invest 1996;97:2753– 62. 12. Caruso RC, Nussenblatt RB, Csaky KG, et al. Assessment of visual function in patients with gyrate atrophy who are considered candidates for gene replacement. Arch Ophthalmol 2001;119:667–9. 13. Liu MM, Tuo J, Chan CC. Gene therapy for ocular diseases. Br J Ophthalmol 2011;95:604 –12. 14. Howden SE, Gore A, Li Z, et al. Genetic correction and analysis of induced pluripotent stem cells from a patient with gyrate atrophy. Proc Natl Acad Sci U S A 2011;108:6537– 42. 15. Wilson DJ, Weleber RG, Green WR. Ocular clinicopathologic study of gyrate atrophy. Am J Ophthalmol 1991;111:24 –33. 16. Meyer CH, Hoerauf H, Schmidt-Erfurth U, et al. Correlation of morphologic changes between optical coherence tomography and topographic angiography in a case of gyrate atrophy [in German]. Ophthalmologe 2000;97:41– 6. 17. Oliveira TL, Andrade RE, Muccioli C, et al. Cystoid macular edema in gyrate atrophy of the choroid and retina: a fluorescein angiography and optical coherence tomography evaluation. Am J Ophthalmol 2005;140:147–9. 18. Vasconcelos-Santos DV, Magalhaes EP, Nehemy MB. Macular edema associated with gyrate atrophy managed with intravitreal triamcinolone: a case report. Arq Bras Oftalmol 2007;70:858 – 61.
Sergouniotis et al 䡠 Retinal Structure and Function in Gyrate Atrophy 19. Sharma YR, Singh DV, Azad RV, Pal N. Gyrate atrophy with bilateral full thickness macular hole [letter]. Eye (Lond) 2006; 20:745–7. 20. Valle DL, Boison AP, Jezyk P, Aguirre G. Gyrate atrophy of the choroid and retina in a cat. Invest Ophthalmol Vis Sci 1981;20:251–5. 21. Mashima YG, Weleber RG, Kennaway NG, Inana G. Genotype-phenotype correlation of a pyridoxine-responsive form of gyrate atrophy. Ophthalmic Genet 1999;20:219 –24. 22. Drexler W, Fujimoto JG. State-of-the-art retinal optical coherence tomography. Prog Retin Eye Res 2008;27:45– 88. 23. Delori FC, Dorey CK, Staurenghi G, et al. In vivo fluorescence of the ocular fundus exhibits retinal pigment epithelium lipofuscin characteristics. Invest Ophthalmol Vis Sci 1995;36:718 –29. 24. Schmitz-Valckenberg S, Holz FG, Bird AC, Spaide RF. Fundus autofluorescence imaging: review and perspectives. Retina 2008;28:385– 409. 25. Rohrschneider K, Bultmann S, Springer C. Use of fundus perimetry (microperimetry) to quantify macular sensitivity. Prog Retin Eye Res 2008;27:536 – 48. 26. Srinivasan VJ, Monson BK, Wojtkowski M, et al. Characterization of outer retinal morphology with high-speed, ultrahigh-resolution optical coherence tomography. Invest Ophthalmol Vis Sci 2008;49:1571–9. 27. Midena E. Perimetry and the Fundus: An Introduction to Microperimetry. Thorofare, NJ: SLACK, Inc.; 2007:160. 28. Kumar P, Henikoff S, Ng PC. Predicting the effects of coding non-synonymous variants on protein function using the SIFT algorithm. Nat Protoc 2009;4:1073– 81. 29. Adzhubei IA, Schmidt S, Peshkin L, et al. A method and server for predicting damaging missense mutations [letter]. Nat Methods 2010;7:248 –9. 30. Zweifel SA, Engelbert M, Laud K, et al. Outer retinal tubulation: a novel optical coherence tomography finding. Arch Ophthalmol 2009;127:1596 – 602. 31. Kaufman DL, Ramesh V, McClatchey AI, et al. Detection of point mutations associated with genetic diseases by an exon scanning technique. Genomics 1990;8:656 – 63.
32. Brody LC, Mitchell GA, Obie C, et al. Ornithine deltaaminotransferase mutations in gyrate atrophy: allelic heterogeneity and functional consequences. J Biol Chem 1992;267:3302–7. 33. Kaiser-Kupfer M, Kuwabara T, Uga S, et al. Cataract in gyrate atrophy: clinical and morphologic studies. Invest Ophthalmol Vis Sci 1983;24:432– 6. 34. Takki K. Differential diagnosis between the primary total choroidal vascular atrophies. Br J Ophthalmol 1974;58:24 –35. 35. Ho G, Kumar S, Min XS, et al. Molecular imaging of retinal gliosis in transgenic mice induced by kainic acid neurotoxicity. Invest Ophthalmol Vis Sci 2009;50:2459 – 64. 36. Fujiwara T, Imamura Y, Giovinazzo VJ, Spaide RF. Fundus autofluorescence and optical coherence tomographic findings in acute zonal occult outer retinopathy. Retina 2010;30:1206 –16. 37. Ma N, Streilein JW. Contribution of microglia as passenger leukocytes to the fate of intraocular neuronal retinal grafts. Invest Ophthalmol Vis Sci 1998;39:2384 –93. 38. de Kok YJ, Vossenaar ER, Cremers CW, et al. Identification of a hot spot for microdeletions in patients with X-linked deafness type 3 (DFN3) 900 kb proximal to the DFN3 gene POU3F4. Hum Mol Genet 1996;5:1229 –35. 39. den Hollander AI, Koenekoop RK, Yzer S, et al. Mutations in the CEP290 (NPHP6) gene are a frequent cause of Leber congenital amaurosis. Am J Hum Genet 2006;79:556 – 61. 40. Ramesh V, McClatchey AI, Ramesh N, et al. Molecular basis of ornithine aminotransferase deficiency in B-6-responsive and -nonresponsive forms of gyrate atrophy. Proc Natl Acad Sci U S A 1988;85:3777– 80. 41. Michaud J, Thompson GN, Brody LC, et al. Pyridoxineresponsive gyrate atrophy of the choroid and retina: clinical and biochemical correlates of the mutation A226V. Am J Hum Genet 1995;56:616 –22. 42. Ohkubo Y, Ueta A, Ito T, et al. Vitamin B6-responsive ornithine aminotransferase deficiency with a novel mutation G237D. Tohoku J Exp Med 2005;205:335– 42.
Footnotes and Financial Disclosures Originally received: June 21, 2011. Final revision: September 7, 2011. Accepted: September 8, 2011. Available online: December 17, 2011.
Manuscript no. 2011-916.
1
Institute of Ophthalmology, University College London, London, United Kingdom.
2
Moorfields Eye Hospital, London, United Kingdom. Supported by The British Retinitis Pigmentosa Society; Fight for Sight; Moorfields Eye Hospital Special Trustees, London, United Kingdom; The National Institute for Health Research UK to the Biomedical Research Centre for Ophthalmology based at Moorfields Eye Hospital NHS Foun-
dation Trust and UCL Institute of Ophthalmology; and The Foundation Fighting Blindness. The sponsors or funding organizations had no role in the design or conduct of this research. Financial Disclosure(s): The author(s) have no proprietary or commercial interest in any materials discussed in this article. Correspondence: Andrew R. Webster, MD, FRCOphth, Institute of Ophthalmology, University College London, 11-43 Bath Street, London EC1V 9EL, United Kingdom. E-mail: [email protected]. *These authors contributed equally as first authors.
605
Gyrate atrophy of the choroid and retina is a genetically determined, progressive condition associated with significantly increased plasma ornithine levels and inherited as an autosomal recessive trait.1 Patients typically report night blindness, loss of peripheral vision, or both in the second decade of life; myopia and early cataracts also are features of the disorder. On funduscopy, sharply demarcated, circular areas of chorioretinal atrophy distributed around the peripheral fundus are observed that, later in the disease process, coalesce and spread posteriorly. The macula and, consequently, central vision often is preserved into the 4th or 5th decade of life.2,3 More than 200 individuals with gyrate atrophy have been reported since it was first described in the late 19th century, with a significant proportion of them being Finnish.4 The underlying biochemical defect of gyrate atrophy is in ornithine aminotransferase (OAT), a vitamin B6-dependant mitochondrial matrix enzyme. Ornithine aminotransferase cat-
596
© 2012 by the American Academy of Ophthalmology Published by Elsevier Inc.
alyzes the conversion of L-ornithine, a nonproteinaceous amino acid, to proline and glutamic acid. Ornithine aminotransferase plays a role in cellular detoxification by disposing ornithine derived from dietary arginine, thus removing uncoupled ammonia.5– 8 Chronic reduction of plasma ornithine levels with an arginine-restricted diet has been reported to slow disease progression in some instances.9 The gene encoding the 439-amino acid human OAT precursor is expressed in most tissues, including liver, brain, neurosensory retina, and retinal pigment epithelium (RPE).10 More than 50 mutations have been reported in this gene (HGMD; http://www.hgmd.cf.ac.uk/; accessed June 2, 2011) with their deleterious consequences largely confined to the eye. Because the RPE is considered the primary site of dysfunction in gyrate atrophy, clinical trials of gene replacement therapy targeting the RPE or cellular therapeutic approaches involving induced pluripotent stem cell-derived RPE may be relevant to gyrate atrophy patients.11–14 ISSN 0161-6420/12/$–see front matter doi:10.1016/j.ophtha.2011.09.017
Sergouniotis et al 䡠 Retinal Structure and Function in Gyrate Atrophy Histopathologic study of postmortem tissue and in vivo cross-sectional imaging with time-domain (TD) optical coherence tomography (OCT) previously were used to provide insight into structural changes in gyrate atrophy.15–19 Retinal histopathologic analysis has been reported in human as well as in animal models of gyrate atrophy.11,15,20 In a 98-year-old patient, later identified to be homozygous for a p.Glu318Lys mutation,21 focal areas of photoreceptor atrophy with an underlying hyperplastic RPE were observed in the posterior pole. Near the transition from relatively preserved to atrophic retina, the RPE was either absent or dysmorphic. More peripherally to this transition zone, both RPE and photoreceptor cells were absent. At a short distance into the area of atrophic RPE, photoreceptor cell nuclei abutted directly onto Bruch’s membrane; the underlying choriocapillaris were preserved. Complete absence of outer retina, RPE, choriocapillaris, and most medium and large choroidal vessels was observed in the atrophic region.15 Regarding in vivo investigation, although resolution is relatively low in TD OCT detection, features such as edema and macular hole have been described.17–19 Spectraldomain (SD) OCT is an alternative to TD OCT with significant sensitivity and speed advantages, allowing highresolution imaging of tissue microstructure.22 Another noninvasive retinal imaging technology is fundus autofluorescence (FAF). Fundus autofluorescence uses naturally occurring fluorescence to provide an indicator of RPE structure and RPE and photoreceptor physiologic features.23,24 To assess the functional significance of abnormalities detected by SD OCT and FAF, testing of retinal function is required. Fundus-controlled microperimetry provides a method for accurate functional assessment of the central retina with high spatial resolution and continuous fixation monitoring.25 The present study describes the retinal structure and function in 7 patients with clinical, genetic, and biochemical evidence of gyrate atrophy. Novel FAF, SD OCT, and microperimetry findings are presented. Four novel mutations in the OAT gene are reported, and monoallelic expression of a mutated allele is demonstrated.
Patients and Methods Patients and Clinical Examination Seven unrelated patients with a diagnosis of gyrate atrophy were ascertained at Moorfields Eye Hospital, London, United Kingdom, over a 10-year period; all had a retinal dystrophy and funduscopic features compatible with those previously described in gyrate atrophy, together with significantly raised plasma ornithine levels. After informed consent was obtained, blood samples were collected and genomic DNA was extracted from peripheral blood leukocytes. The study was approved by the Moorfields and Whittington Hospitals local ethics committee, and all investigations were conducted in accordance with the principles of the Declaration of Helsinki. Patients 1 through 7 are numbered according to age, youngest to oldest. Clinical assessment included best-corrected visual acuity testing using the Early Treatment Diabetic Retinopathy Study charts, dilated fundus examination, color fundus photography (TRC-50IA; Topcon, Tokyo, Japan), FAF, SD OCT, and
fundus-controlled static microperimetry. The Spectralis HRA⫹OCT with viewing module version 5.1.2.0 (Heidelberg Engineering, Heidelberg, Germany) was used to acquire FAF images (over a 30⫻30° field, a 55⫻55° field, or both; 6 patients tested) and tomographs (all 7 patients tested). The SD OCT protocol included a horizontal, linear scan (100 B-scans averaged to improve the signal-to-noise ratio) centered on the fovea and a volume scan (minimum of 19 B-scan slices, 20⫻20° or 20⫻15°) for each eye. Retinal thickness in the central and inner macula Early Treatment Diabetic Retinopathy Study subfields were assessed; HEYEX software interface (version 1.6.2.0; Heidelberg Engineering) was used for all measurements. Outer retina morphologic feature interpretation was as suggested by Srinivasan et al.26 Static microperimetry (MP1 Microperimeter; Nidek Technologies, Padova, Italy) was performed in 6 patients. The semiautomatic examination with the number of stimuli ranging from 49 through 82, threshold sensitivities from 0 through 20 dB, test spot size Goldmann I, projection time of 1000 ms, and 4-2 staircase test strategy was used. A smaller than usual stimulus size was chosen because it previously was suggested that with such stimuli, it is possible to observe little islands with residual function.27
DNA Sequencing, mRNA Analysis, and Bioinformatics All 9 coding exons and flanking intronic boundaries of OAT were analyzed by direct sequencing of polymerase chain reaction (PCR) amplicons using the primers and conditions listed in Table 1 (available at http://aaojournal.org). When possible, parental DNA samples were screened to establish the phase of variants (patients 4 and 6). Leukocyte RNA was extracted from whole blood using a QIAamp RNA blood extraction kit (Qiagen, Crawley, United Kingdom) according to the manufacturer’s guidelines. cDNA was reverse-transcribed using a cDNA synthesis kit (BioLine, London, United Kingdom) with a random hexamer primer mix. For reversetranscriptase (RT) PCR reactions, OAT was amplified between exons 2 and 9 and between exons 6 and 10, respectively. The housekeeping gene -actin also was amplified as a positive control. Details of RT PCR primers used are listed in Table 2 (available at http://aaojournal.org). The identity of OAT RT PCR products was established by direct sequencing using standard procedures. Two different bioinformatics tools were applied to predict the effect of amino acid substitutions on OAT function: SIFT (version 4.0.3; available at http://sift.jcvi.org/; accessed June 2, 2011),28 which uses sequence homology to make predictions, and PolyPhen-2 (version 2.0.23; available at http://genetics.bwh.harvard.edu/pph2/; accessed June 2, 2011), which uses both sequence-based and structure-based predictive features.29
Results Clinical Findings Table 3 presents data on the 7 patients studied (age range, 10 –52 years). The mean visual acuity was 0.26 logarithm of the minimum angle of resolution units (range, 0.10 – 0.50 logarithm of the minimum angle of resolution units). Presentation was in the first decades of life: 4 patients were noted to have abnormal retinal appearance in routine refraction after difficulty reading the blackboard in early school years (patients 1, 2, 4, and 6); 1 patient had night vision problems at approximately 12 years of age (patient 5); 1 patient recalled night blindness from a young age and problems
597
Ophthalmology Volume 119, Number 3, March 2012 Table 3. Clinical Characteristics and Molecular Pathologic
Refractive Error (Diopters) Patient No.
Gender
Age at Examination (yrs)
1 2 3 4 5 6 7
F F F M F F F
10 14 17 30 34 38 52
Right Eye
Left Eye
⫺6.75/⫺4.00⫻180* ⫺6.50/⫺4.00⫻180* ⫺6.00/⫺2.50⫻25 ⫺5.50/⫺2.50⫻155 ⫺2.00 ⫺1.75/⫺0.50⫻170 Myopic astigmatism ⫺13.50/⫺1.50⫻75† ⫺11.00/⫺1.50⫻120† Myopic astigmatism† ⫺9.50/⫺2.00⫻177† ⫺7.75/⫺3.00⫻7†
Visual Acuity (Logarithm of the Minimum Angle of Resolution) Right Eye
Left Eye
0.34 0.10 0.22 0.18 0.50 0.10 0.50
0.30 0.16 0.20 0.20 0.20 0.40 0.20
F ⫽ female; HRR ⫽ Hardy-Rand-Rittler device; ISH ⫽ Ishihara color vision test; M ⫽ male. Color vision was evaluated using the HRR (American Optical Company, New York, NY) with both eyes open or the 24-plate ISH (Kanehara and Co. *Cycloplegic refraction. † Prior cataract surgery. ‡ Diagnosed with retinal dystrophy; gyrate atrophy was diagnosed at age 30 years.
with peripheral vision from early teenage years, but medical advice was sought only at age 26 years (patient 7); and 1 patient had poor central vision (1.00 logarithm of the minimum angle of resolution units in each eye) with electrophysiology, suggesting that this was the result of functional visual loss. Six of 7 patients were night blind, and 7 of 7 had elevated plasma ornithine concentration. Patients 5 and 7 underwent bilateral cataract extractions with intraocular lens implants at 29 and 41 years of age, respectively; patient 6 underwent surgery in the right eye operated at 22 years of age and in the left eye at 26 years of age. Patient 7 had late-onset sensorineural hearing loss and a paternal family history of hearing impairment; hearing problems also were reported for patient 1. All patients had a negative family history of retinal disease; patients 1 and 2 were born to consanguineous parents. Funduscopy revealed typical changes of gyrate atrophy in all 7 patients. Importantly, retinal pigment migration was minimal or absent and retinal vessel diameter was relatively preserved. Sharply demarcated areas of increased or preserved signal were observed in FAF images of all 6 cases tested. Color fundus photography and FAF imaging are presented in Figure 1. Spectral-domain OCT demonstrated structural changes that were concordant between eyes in all patients. For patients 1 and 3, SD OCT revealed multiple intraretinal cystic spaces bilaterally and no other significant abnormality (Fig 2). Hyporeflective cysts varying in size also were detected in the remaining 5 cases. In these patients (patients 2, 4, 5, 6, and 7), the hyperreflective band corresponding to the photoreceptor inner segment and outer segment (IS/OS) junction was preserved in the regions where autofluorescence signal was normal or increased. Outside of these regions, typically at the edges of foveal scans and at the more peripheral scans of the volume series, extensive loss of IS/OS junction reflectivity and significant thinning were observed. The thinning was not confined to the scattering layers in the outer retina, but also extended to the inner retinal layers, the hyperreflective band corresponding to the RPE–Bruch’s complex, and the structures often seen posterior to it. Structures corresponding to the external choroidal margins, large choroidal vessels, and inner scleral surface were visible on the scans of all patients. A loss of focal hyperreflectivity in the inner choroid was observed frequently, and structures resembling large choroidal vessels appeared to be in close proximity to the hyperreflective band interpreted as Bruch’s membrane. Vitreomacular traction, epiretinal membrane, or both, causing irregularities of the retinal surface, were observed in the 4 oldest patients. An interesting shared
598
feature in the SD OCT images of all 7 patients was the presence of hyperreflective deposit in the ganglion cell layer, particularly over the regions of preserved autofluorescence or at a short distance into the regions of absent autofluorescence. When the amount of hyperreflective material was increased, deposits coalesced and were depicted as vertical hyperreflective lines (for example Fig 2, patients 3 and 4; Fig 3B, patient 4, available at http://aaojournal.org). Another interesting finding was the presence of round or ovoid hyporeflective spaces with hyperreflective borders on SD OCT scans from the 4 older patients (patients 4, 5, 6, and 7). These typically were found in the transition from relatively preserved to atrophic retina (Fig 4A, patient 4) and previously were termed outer retinal tubulation.30 Finally, when thickness profiles were evaluated, relative thickening was observed in the more central Early Treatment Diabetic Retinopathy Study subfields of younger patients (Fig 3A, patients 1, 2, and 3, available at http://aaojournal.org). Static microperimetry testing results were overlaid with FAF and SD OCT and are presented in Figure 4. Stable fixation was observed in all eyes tested. In patients with structurally preserved posterior pole, although appropriate normative data are lacking, a mild reduction in macular sensitivity is likely (Fig 4A-B, patients 1 and 3). In the more severely affected individuals (patients 4 through 7), reduced sensitivity was evident over regions of increased or preserved autofluorescence signal (on FAF) and relatively intact IS/OS junction line (on SD OCT). In 6 of 8 eyes, outside these areas, patients did not respond even to the brightest stimuli (Fig 4A-B, patients 4, 5, 6, and 7). Interestingly, in the remaining 2 eyes, consistent responses were documented over areas with significant outer retinal loss (absent IS/OS junction line on SD OCT) and RPE loss (no signal on FAF; Fig 4C, patients 4 and 7).
Molecular Findings Overall, 9 different likely disease-causing DNA variants in OAT were detected in 13 alleles of 7 patients and included 7 missense and 2 nonsense changes (Table 3). The predicted effects of each of the 7 amino acid substitutions are summarized in Table 4.21,31,32 The bioinformatics programs SIFT and PolyPhen-2 both supported the likely pathogenicity of each of the identified missense mutations, including p.Gly51Asp, p.Gly91Glu, and p.Gly121Asp, which are novel to this study. Furthermore, all novel mutations identified (p.Gly51Asp, p.Gly91Glu, p.Gly121Asp, and p.Arg250X) were found to be absent from the
Sergouniotis et al 䡠 Retinal Structure and Function in Gyrate Atrophy Features of Patients with Gyrate Atrophy
Color Vision
Presenting Symptom (Age at Diagnosis [yrs])
Nyctalopia (Age First Noted [yrs])
Ornithine Levels at Diagnosis (mol/l)
Molecular Diagnosis, Mutations in OAT
HRR full HRR full ISH 17/17 ISH 17/17 ISH test plate only ISH 6/17 ISH 5/17
Increasing myopia (9) Increasing myopia (11) Poor central vision (17) Increasing myopia (6‡) Night blindness (13) Increasing myopia (9) Night blindness (26)
Yes (8) No Yes (17) Yes (14) Yes (12) Yes (27) Yes (always)
1390 1152 775 908 ⬃900 ⬃1000 ⬃700
p.[Gly91Glu];[Gly91Glu] p.[Gly51Asp];[Gly51Asp] p.[Glu318Lys];[?] p.[Pro199Gln];[Pro417Leu] p.[Pro199Gln(;)Pro417Leu] p.[Gly121Asp];[Tyr299X] p.[Tyr55His(;)Arg250X]
Ltd, Tokyo, Japan) with each eye; the best score is presented. Normal plasma ornithine levels: 20 to 114 mol/l. Novel changes are in boldface.
1000 genomes project data set (628 individuals from the 20100804 sequence and alignment release). Because patient 3 was identified as harboring only a single heterozygous missense mutation in OAT (p.Glu318Lys), patient leukocyte mRNA was analyzed by RT PCR. Two pairs of RT PCR primers were designed to amplify between OAT exons 2 through 9 and between exons 6 through 10, respectively (Table 2, available at http://aaojournal.org). The PCR products of the expected sizes (970 bp and 596 bp) were amplified for both control and patient samples (Fig 5A, available at http://aaojournal.org). Direct sequencing of the RT PCR products demonstrated the patient apparently to be homozygous for the likely pathogenic p.Glu318Lys variant, which previously was identified in the heterozygous state by direct sequencing of genomic DNA. In addition, RT PCR products revealed apparent homozygosity for a single nucleotide polymorphism (c.1134C¡T, p.(⫽); rs11461), which had been identified in the heterozygous state when sequencing the patient’s genomic DNA (Fig 5B, available at http://aaojournal.org). These data strongly suggest monoallelic expression of the disease-causing allele (p.Glu318Lys), which is likely to be the result of an unidentified sequence variant abrogating expression from one of the patient’s chromosomes (Fig 5C, available at http://aaojournal.org).
Discussion This report describes retinal structure and function in 7 gyrate atrophy patients and reports 4 novel mutations. Novel data related to imaging studies (FAF and SD OCT) are presented, providing insights into the anatomic changes at both early and late stages of gyrate atrophy. Microperimetry testing results are reported, and the relationship between structural abnormalities and functional deficits is evaluated. All 7 study patients had myopic refraction, fundus changes typical of gyrate atrophy, and greatly elevated plasma ornithine levels. Six of 7 patients reported poor night
vision, increasing myopia, or both as their first symptom(s). In a previous report, more than 70% of subjects presented with increasing myopia at presentation.4 Funduscopy through dilated pupils in highly myopic children with or without night blindness is key for early diagnosis of gyrate atrophy. However, it is noted that presentation can be unusual, as in patient 3, an individual with low myopia with poor central vision, probably unrelated to the primary retinopathy. Visual acuity loss in patients with gyrate atrophy can be the result of disease progression causing central photoreceptor cell death, but also cataract or macular edema. By the second decade, cataract is considered a uniform finding in gyrate atrophy patients.33 Cataracts typically are posterior sutural, may have unique histologic characteristics, and require surgical intervention by the third or fourth decade of life.3,33 In the present series, the 3 older patients (patients 5, 6, and 7) already had undergone bilateral cataract surgery. Cystoid macular edema can be another ocular manifestation of gyrate atrophy, with at least 4 cases reported to date; fluorescein angiography and TD OCT have been used to confirm this macular complication.17,18 Hyporeflective cystic spaces, predominantly at the inner retina, were identified with SD OCT in all patients, suggesting that edema also is a uniform finding. Retinal pigment epithelial dysfunction or loss leading to disruption of the outer blood–retinal barrier and diffusion of fluid toward intraretinal spaces is likely to be the main mechanism of edema formation in gyrate atrophy. The fact that it is observed even in younger patients with structurally intact, although thickened (Fig 3, available at http://aaojournal.org), retina may suggest RPE dysfunction as an early abnormality leading to photoreceptor cell exposure to toxic agents from choroidal circulation. In older patients, in addition to the inner retinal hyporeflective cystic
™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™3 Figure 1. Color fundus photography and fundus autofluorescence (FAF) imaging of patients with gyrate atrophy and OAT mutations. The number relating to each pair of images corresponds to the case number (patients 1 through 7) and patients are numbered according to age (youngest to oldest; age range, 10 –52 years). Bone spicule pigmentation is minimal, retinal vessels are relatively preserved, and circular areas of chorioretinal atrophy are evident in fundus photographs. Sharply demarcated areas of increased or normal signal are observed in FAF images.
599
Ophthalmology Volume 119, Number 3, March 2012
600
Figure 2. Foveal linear spectral-domain optical coherence tomography scans of 7 patients with gyrate atrophy (patients 1 through 7, ordered by age).
601
Ophthalmology Volume 119, Number 3, March 2012
602
Sergouniotis et al 䡠 Retinal Structure and Function in Gyrate Atrophy Table 4. Missense Coding Sequence Changes in OAT That Are Likely to be Pathogenic and In Silico Analysis of Their Effect SIFT
Polyphen-2
Nucleotide Substitution
Amino Acid Exchange
Prediction
Tolerance Index (0–1)
Prediction
HumVar Score (0–1)
Reference
c.152 G¡A c.163 T¡C c.272 G¡A c.362 G¡A c.596 C¡A c.952 G¡A c.1250 C¡T
p.Gly51Asp p.Tyr55His p.Gly91Glu p.Gly121Asp p.Pro199Gln p.Glu318Lys p.Pro417Leu
Intolerant Intolerant Intolerant Intolerant Intolerant Intolerant Intolerant
0.00 0.00 0.00 0.01 0.00 0.02 0.00
POS PRD PRD PRD PRD POS PRD
0.867 0.980 1.000 0.995 0.994 0.626 0.997
This study Kaufman et al., 199031 This study This study Kaufman et al., 199031 Mashima et al., 199921 Brody et al., 199232
POS ⫽ possibly damaging; PRD ⫽ probably damaging; SIFT ⫽ sorting intolerant from tolerant (http://sift.jcvi.org/). SIFT results are reported to be tolerant if the tolerance index is ⱖ0.05 or intolerant if tolerance index is ⬍0.05. Polyphen-2 (version 2.1.0) appraises mutations qualitatively as benign, POS, or PRD based on the model’s false-positive rate. The cDNA is numbered according to Ensembl transcript ID ENST00000368845, in which ⫹1 is the A of the translation start codon.
spaces, SD OCT detected epiretinal membrane and vitreomacular traction. Hyperreflective deposits on SD OCT were observed in the ganglion cell layer of all gyrate atrophy patients in this report (Fig 2, patients 3 and 4; Fig 3B, patient 4, available at http://aaojournal.org). This material was more obvious but not confined to the areas near the transition from relatively preserved to atrophic retina. Colorless, elongated, glittering crystals, better seen in the fluorescein angiogram and located over or proximal to the preserved areas, have been described previously in gyrate atrophy patients.2,34 Immunofluorescent localization of glial fibrillary acidic protein in the Oat–/– mouse retina revealed labeling of the astrocytes in the innermost retina.11 This finding suggests gliotic response to ongoing cell death,35 and one possibility is that the hyperreflective deposits on SD OCT reflect retinal gliosis. Inner retinal globular hyperreflective aggregates, with some similarities to those described in this study, previously were reported in eyes with acute zonal occult outer retinopathy.36 Another interesting finding in the tomographs of older gyrate atrophy patients was the presence of outer retinal tubulation (Fig 4, patient 4). Similar structures have been described previously in Bietti crystalline dystrophy, pseudoxanthoma elasticum, and age-related macular degeneration.30 It has been proposed that these rosettes are formed by degenerating photoreceptor cells becoming arranged in a
circular or ovoid fashion.30 This may explain the relative preservation of visual function over these structures (Fig 4, patient 4). Because RPE is an early site of dysfunction in all conditions mentioned above, the authors propose that the first step in the process may be RPE loss. In support of this is evidence from transplantation studies in murine eyes, which suggests that symbiosis of photoreceptors with microglia in rosettes promotes photoreceptor cell survival in the absence of RPE.37 This study showed that in gyrate atrophy patients, microperimetry stimuli are recognized over areas of preserved or increased signal in FAF. Surprisingly, in 2 eyes of 2 patients (Fig 4B), preserved retinal function also was observed over areas of absent autofluorescence signal, corresponding to RPE atrophy. When the relevant microperimetry data points were overlaid with SD OCT, a very thin retina with complete loss of the IS/OS junction line was seen (Fig 4C). It can be speculated that reflection, or scatter, of the stimulus from the sclera onto preserved and functioning anterior retina may account for the absence of an absolute scotoma, which would be expected from an area of complete loss of RPE and outer retina. Of the 2 eyes concerned, the results were reproducible and not evidently the result of poor compliance. When the patients were asked to report the direction of the stimuli, again this was reported reproducibly, but did not always map to the area stimulated. Each eye had areas of intact anterior retina, and the most
4™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™ Figure 4. Functional assessment of the central retina in 6 patients with gyrate atrophy. A, Static microperimetry testing results overlaid with fundus autofluorescence (FAF) images and spectral-domain optical coherence tomography (SD OCT) scans from patient 3 (17 years of age) and patient 4 (30 years of age) are presented. Mild reduction of foveal sensitivity is observed in the younger patient, for whom the FAF image shows normal results and the SD OCT scan shows only some cystic spaces. In the older patient, sensitivity is reduced over areas of relatively preserved FAF and with an intact inner segment– outer segment junction line. However, outside these regions, there is no response even to the brightest stimulus. The asterisk marks a round hyporeflective space with hyperreflective borders (retinal tubulation). This may reflect photoreceptors forming rosettes in the absence of retinal pigment epithelium. B, Static microperimetry results from patient 1 (10 years of age), patient 5 (34 years of age), patient 6 (38 years of age), and patient 7 (52 years of age). Data sets have been overlaid with infrared (patient 1) or FAF (patients 5, 6, and 7) images. Similarly to patient 3, patient 1 has mildly reduced retinal sensitivity despite structurally intact retina. Conversely, in the older patients, the retina is functional only over areas of preserved autofluorescence, thus highlighting the clinical value of FAF in gyrate atrophy. C, Static microperimetry testing results overlaid with FAF images and SD OCT scans from patient 4 (30 years of age) and patient 7 (52 years of age) are presented. Surprisingly, retinal function appears to be preserved over areas of absent autofluorescence signal (on FAF) and very thin outer and total retina (on SD OCT). The scleral surface is clearly visible, and the presence of islands of preserved photoreceptors cannot be excluded (arrowhead).
603
Ophthalmology Volume 119, Number 3, March 2012 likely explanation is that scatter toward the functioning areas allowed the patient to see these stimuli. Alternative explanations include small areas of preserved retina missed by OCT and FAF imaging, and inner-retinal phototransduction. This finding suggests the need for caution in the interpretation of microperimetry data when investigating dystrophies such as gyrate atrophy, and is particularly relevant to its use in characterizing the deficit in choroideremia in preparation for relevant therapeutic trials. Six of 7 patients in this study were identified as harboring either proven (patients 1, 2, 4, and 6) or presumed (patients 5 and 7) biallelic mutations in OAT. Patients 4 and 5, who are not knowingly related, share the same genotype. The observed phenotypic differences between the 2 patients most likely can be attributed to other currently unidentified genetic factors, environmental modifying factors, or both. Patient 3 was found to harbor only a single previously reported21 heterozygous OAT mutation, p.Glu318Lys. Interestingly, patient 3 is of British ancestry, and all previously reported individuals carrying this mutation also are of British or Irish descent.21 Because gyrate atrophy is a recessive condition caused by biallelic mutations, the patient’s leukocyte mRNA was analyzed by RT PCR to establish whether OAT allelic expression was abnormal. Although the correctly sized RT PCR products were amplified from the patient sample, direct sequencing of the amplimers demonstrated monoallelic expression of the disease-causing allele (Fig 4). The underlying cause of this loss of expression has not been identified; however, it may represent disruption of the OAT promoter, mutation of a regulatory element potentially distant from the coding sequence, or an undetected intronic mutation.38,39 This finding demonstrates that it is possible to amplify OAT mRNA from peripheral blood leucocytes, and thus to investigate potential alterations in pre-mRNA splicing, expression of OAT for diagnostic purposes, or both. In addition, this has important implications for future gyrate atrophy diagnostic studies because OAT allelic gene expression analysis has the potential to indicate the presence of mutations that will be missed by conventional mutation detection methods. The Glu318 residue is important for pyridoxal phosphate (the active form of vitamin B6) cofactor binding to OAT,7 and it has been suggested previously that a p.Glu318Lys substitution disrupts it.15,21 This missense change is one of only a handful of OAT mutations demonstrated to be responsive to pyridoxal phosphate supplementation.21,40 – 42 Importantly, gyrate atrophy patients reported with pyridoxal phosphate-responsive genotypes have been noted to have a less severe and progressive clinical presentation,21 a finding in keeping with the clinical presentation of patient 3 in this study. In conclusion, the present study expands our knowledge of gyrate atrophy. Structural changes are reported in detail: macular edema is a common finding, the fovea is relatively thick in early stages of disease, deposits that may suggest retinal gliosis are observed, and retinal tubulation is present in advanced disease. Functional testing highlights the importance of FAF as a monitoring tool for gyrate atrophy and suggests that retinal sensitivity can be reduced even over structurally intact retina and in early stages of disease pro-
604
gression. Allelic heterogeneity is high in the disorder, and leukocyte RNA analysis can increase the sensitivity of mutation detection.
References 1. Simell O, Takki K. Raised plasma-ornithine and gyrate atrophy of the choroid and retina. Lancet 1973;1:1031–3. 2. Takki K. Gyrate atrophy of the choroid and retina associated with hyperornithinaemia. Br J Ophthalmol 1974;58:3–23. 3. Takki KK, Milton RC. The natural history of gyrate atrophy of the choroid and retina. Ophthalmology 1981;88:292–301. 4. Peltola KE, Nanto-Salonen K, Heinonen OJ, et al. Ophthalmologic heterogeneity in subjects with gyrate atrophy of choroid and retina harboring the L402P mutation of ornithine aminotransferase. Ophthalmology 2001;108:721–9. 5. O’Donnell JJ, Sandman RP, Martin SR. Gyrate atrophy of the retina: inborn error of L-ornithin:2-oxoacid aminotransferase. Science 1978;200:200 –1. 6. Volpe P, Sawamura R, Strecker HJ. Control of ornithin deltatransaminase in rat liver and kidney. J Biol Chem 1969;244: 719 –26. 7. Shen BW, Hennig M, Hohenester E, et al. Crystal structure of human recombinant ornithine aminotransferase. J Mol Biol 1998;277:81–102. 8. Valle D, Walser M, Brusilow SW, Kaiser-Kupfer M. Gyrate atrophy of the choroid and retina: amino acid metabolism and correction of hyperornithinemia with an arginine-deficient diet. J Clin Invest 1980;65:371– 8. 9. Kaiser-Kupfer MI, Caruso RC, Valle D. Gyrate atrophy of the choroid and retina: further experience with long-term reduction of ornithine levels in children. Arch Ophthalmol 2002; 120:146 –53. 10. Rao GN, Cotlier E. Ornithine delta-aminotransferase activity in retina and other tissues. Neurochem Res 1984;9:555– 62. 11. Wang T, Milam AH, Steel G, Valle D. A mouse model of gyrate atrophy of the choroid and retina: early retinal pigment epithelium damage and progressive retinal degeneration. J Clin Invest 1996;97:2753– 62. 12. Caruso RC, Nussenblatt RB, Csaky KG, et al. Assessment of visual function in patients with gyrate atrophy who are considered candidates for gene replacement. Arch Ophthalmol 2001;119:667–9. 13. Liu MM, Tuo J, Chan CC. Gene therapy for ocular diseases. Br J Ophthalmol 2011;95:604 –12. 14. Howden SE, Gore A, Li Z, et al. Genetic correction and analysis of induced pluripotent stem cells from a patient with gyrate atrophy. Proc Natl Acad Sci U S A 2011;108:6537– 42. 15. Wilson DJ, Weleber RG, Green WR. Ocular clinicopathologic study of gyrate atrophy. Am J Ophthalmol 1991;111:24 –33. 16. Meyer CH, Hoerauf H, Schmidt-Erfurth U, et al. Correlation of morphologic changes between optical coherence tomography and topographic angiography in a case of gyrate atrophy [in German]. Ophthalmologe 2000;97:41– 6. 17. Oliveira TL, Andrade RE, Muccioli C, et al. Cystoid macular edema in gyrate atrophy of the choroid and retina: a fluorescein angiography and optical coherence tomography evaluation. Am J Ophthalmol 2005;140:147–9. 18. Vasconcelos-Santos DV, Magalhaes EP, Nehemy MB. Macular edema associated with gyrate atrophy managed with intravitreal triamcinolone: a case report. Arq Bras Oftalmol 2007;70:858 – 61.
Sergouniotis et al 䡠 Retinal Structure and Function in Gyrate Atrophy 19. Sharma YR, Singh DV, Azad RV, Pal N. Gyrate atrophy with bilateral full thickness macular hole [letter]. Eye (Lond) 2006; 20:745–7. 20. Valle DL, Boison AP, Jezyk P, Aguirre G. Gyrate atrophy of the choroid and retina in a cat. Invest Ophthalmol Vis Sci 1981;20:251–5. 21. Mashima YG, Weleber RG, Kennaway NG, Inana G. Genotype-phenotype correlation of a pyridoxine-responsive form of gyrate atrophy. Ophthalmic Genet 1999;20:219 –24. 22. Drexler W, Fujimoto JG. State-of-the-art retinal optical coherence tomography. Prog Retin Eye Res 2008;27:45– 88. 23. Delori FC, Dorey CK, Staurenghi G, et al. In vivo fluorescence of the ocular fundus exhibits retinal pigment epithelium lipofuscin characteristics. Invest Ophthalmol Vis Sci 1995;36:718 –29. 24. Schmitz-Valckenberg S, Holz FG, Bird AC, Spaide RF. Fundus autofluorescence imaging: review and perspectives. Retina 2008;28:385– 409. 25. Rohrschneider K, Bultmann S, Springer C. Use of fundus perimetry (microperimetry) to quantify macular sensitivity. Prog Retin Eye Res 2008;27:536 – 48. 26. Srinivasan VJ, Monson BK, Wojtkowski M, et al. Characterization of outer retinal morphology with high-speed, ultrahigh-resolution optical coherence tomography. Invest Ophthalmol Vis Sci 2008;49:1571–9. 27. Midena E. Perimetry and the Fundus: An Introduction to Microperimetry. Thorofare, NJ: SLACK, Inc.; 2007:160. 28. Kumar P, Henikoff S, Ng PC. Predicting the effects of coding non-synonymous variants on protein function using the SIFT algorithm. Nat Protoc 2009;4:1073– 81. 29. Adzhubei IA, Schmidt S, Peshkin L, et al. A method and server for predicting damaging missense mutations [letter]. Nat Methods 2010;7:248 –9. 30. Zweifel SA, Engelbert M, Laud K, et al. Outer retinal tubulation: a novel optical coherence tomography finding. Arch Ophthalmol 2009;127:1596 – 602. 31. Kaufman DL, Ramesh V, McClatchey AI, et al. Detection of point mutations associated with genetic diseases by an exon scanning technique. Genomics 1990;8:656 – 63.
32. Brody LC, Mitchell GA, Obie C, et al. Ornithine deltaaminotransferase mutations in gyrate atrophy: allelic heterogeneity and functional consequences. J Biol Chem 1992;267:3302–7. 33. Kaiser-Kupfer M, Kuwabara T, Uga S, et al. Cataract in gyrate atrophy: clinical and morphologic studies. Invest Ophthalmol Vis Sci 1983;24:432– 6. 34. Takki K. Differential diagnosis between the primary total choroidal vascular atrophies. Br J Ophthalmol 1974;58:24 –35. 35. Ho G, Kumar S, Min XS, et al. Molecular imaging of retinal gliosis in transgenic mice induced by kainic acid neurotoxicity. Invest Ophthalmol Vis Sci 2009;50:2459 – 64. 36. Fujiwara T, Imamura Y, Giovinazzo VJ, Spaide RF. Fundus autofluorescence and optical coherence tomographic findings in acute zonal occult outer retinopathy. Retina 2010;30:1206 –16. 37. Ma N, Streilein JW. Contribution of microglia as passenger leukocytes to the fate of intraocular neuronal retinal grafts. Invest Ophthalmol Vis Sci 1998;39:2384 –93. 38. de Kok YJ, Vossenaar ER, Cremers CW, et al. Identification of a hot spot for microdeletions in patients with X-linked deafness type 3 (DFN3) 900 kb proximal to the DFN3 gene POU3F4. Hum Mol Genet 1996;5:1229 –35. 39. den Hollander AI, Koenekoop RK, Yzer S, et al. Mutations in the CEP290 (NPHP6) gene are a frequent cause of Leber congenital amaurosis. Am J Hum Genet 2006;79:556 – 61. 40. Ramesh V, McClatchey AI, Ramesh N, et al. Molecular basis of ornithine aminotransferase deficiency in B-6-responsive and -nonresponsive forms of gyrate atrophy. Proc Natl Acad Sci U S A 1988;85:3777– 80. 41. Michaud J, Thompson GN, Brody LC, et al. Pyridoxineresponsive gyrate atrophy of the choroid and retina: clinical and biochemical correlates of the mutation A226V. Am J Hum Genet 1995;56:616 –22. 42. Ohkubo Y, Ueta A, Ito T, et al. Vitamin B6-responsive ornithine aminotransferase deficiency with a novel mutation G237D. Tohoku J Exp Med 2005;205:335– 42.
Footnotes and Financial Disclosures Originally received: June 21, 2011. Final revision: September 7, 2011. Accepted: September 8, 2011. Available online: December 17, 2011.
Manuscript no. 2011-916.
1
Institute of Ophthalmology, University College London, London, United Kingdom.
2
Moorfields Eye Hospital, London, United Kingdom. Supported by The British Retinitis Pigmentosa Society; Fight for Sight; Moorfields Eye Hospital Special Trustees, London, United Kingdom; The National Institute for Health Research UK to the Biomedical Research Centre for Ophthalmology based at Moorfields Eye Hospital NHS Foun-
dation Trust and UCL Institute of Ophthalmology; and The Foundation Fighting Blindness. The sponsors or funding organizations had no role in the design or conduct of this research. Financial Disclosure(s): The author(s) have no proprietary or commercial interest in any materials discussed in this article. Correspondence: Andrew R. Webster, MD, FRCOphth, Institute of Ophthalmology, University College London, 11-43 Bath Street, London EC1V 9EL, United Kingdom. E-mail: [email protected]. *These authors contributed equally as first authors.
605