Downloaded from http://bjo.bmj.com/ on November 15, 2016 - Published by group.bmj.com Clinical science Preserved visual function in retinal dystrophy due to hypomorphic RPE65 mutations Sarah Hull,1,2 Graham E Holder,1,2 Anthony G Robson,1,2 Rajarshi Mukherjee,1,2 Michel Michaelides,1,2 Andrew R Webster,1,2 Anthony T Moore1,2,3

▸ Additional material is ABSTRACT later in childhood, possibly as a result of abnormal published online only. To view Background/aims To present detailed phenotypic and accumulation of retinyl esters.67Retinal imaging please visit the journal online fi (http://dx.doi.org/10.1136/ molecular ndings in four patients from four families reveals a variable thinning of the outer nuclear bjophthalmol-2015-308019). with atypical, mild, recessive RPE65-related retinal layer on optical coherence tomography (OCT) and dystrophy and discuss potential implications for gene a characteristically low signal on fundus autofluor- 89 1 replacement therapy. escence (FAF) imaging. Electrophysiology UCL Institute of Methods Four patients from four families with early demonstrates absent rod function but there may be Ophthalmology, London, UK 510 2Moorfields Hospital, onset retinal dystrophy underwent clinical examination, residual cone function in childhood. This may London, UK retinal imaging and electrophysiological testing. reflect the alternative source of 11-cis-retinol that 3Department of Bidirectional Sanger sequencing of all exons and intron– cones obtain from Müller cells.11 Gene therapy Ophthalmology, University of exon boundaries of RPE65 was performed. trials using subretinally administered recombinant California, San Francisco RPE65 Medical Centre, California, Results All patients presented with in early adeno-associated viral vectors expressing USA childhood but demonstrated a mild phenotype with good have so far achieved promising, although largely – visual acuity until at least 19 years of age. All had unsustained improvements in retinal function.12 14 Correspondence to generalised retinal dysfunction on electroretinography. Previous reports of atypical recessive Professor Tony Moore, Central macular thickness on optical coherence RPE65-related disease have described mild pheno- Ophthalmology Department, UCSF School of Medicine, tomography was preserved in those patients with good types due to presumed hypomorphic alleles; a Koret Vision Centre 10 Koret visual acuity. One patient had extensive white dots single patient with a fundus albipunctatus pheno- – Way, San Francisco, throughout the reminiscent of fundus type has also been reported.41518 This present CA 94143, USA; albipunctatus with electrophysiological evidence of study reports further subjects with mild phenotypes [email protected] partial recovery of rod function after prolonged dark and discusses potential mechanisms. Oral presentation at the adaptation. Sanger sequencing identified RPE65 International Society for mutations in all patients including three missense METHODS Genetic Eye Diseases and variants likely to represent hypomorphic alleles. The study protocol adhered to the tenets of the Retinoblastoma biannual Conclusions Hypomorphic mutations of RPE65 are Declaration of Helsinki and received approval from meeting, Halifax, Canada, August 2015. associated with mild disease in childhood with the local ethics committee. Written, informed preservation of good visual acuity into adulthood; they consent was obtained from all participants prior to Received 26 October 2015 may in rare cases be associated with a flecked retina their inclusion in this study. Revised 12 January 2016 appearance similar to fundus albipunctatus. The Patients were ascertained from the inherited Accepted 25 January 2016 fi Published Online First presence of normal visual acuity in patients with retinal disease clinics at Moor elds Eye Hospital. 23 February 2016 hypomorphic mutations in RPE65 suggests that efficiency Each patient underwent a full clinical examination of transduction may not be the limiting factor in including VA and dilated fundus examination. improving visual acuity in trials of gene replacement Retinal fundus imaging was obtained by 35° colour therapy. Rather, it suggests that for optimal recovery of fundus photography (Topcon Great Britain, visual acuity gene replacement therapy may need to be Berkshire, UK); 30 or 55° FAF imaging (Spectralis, given much earlier in childhood. Heidelberg Engineering, Heidelberg, Germany) and either Stratus OCT (patient 2, Carl Zeiss Meditec, Dublin, California, USA) or Spectralis OCT. A full INTRODUCTION field electroretinogram (ERG) and a pattern ERG RPE65 (retinal pigment epithelium-specific protein, (PERG) were obtained in three patients using gold MIM# 180069) encodes a 65 kD visual cycle foil electrodes to incorporate the International protein, retinoid isomerohydrolase, a vital compo- Society for Clinical Electrophysiology of Vision nent of the visual cycle.1 Recessive mutations in (ISCEV) standards; ERG recording in patient 3 was RPE65 are associated with severe early onset retinal performed elsewhere at age 3 with surface electro- dystrophy including Leber congenital amaurosis des.19 20 Suprathreshold binocular (driving) Ester- (LCA) and account for approximately 11% of early man visual fields and uniocular Humphrey 24-2 and – onset rod–cone dystrophy (RCD).2 4 Patients 30-2 threshold visual fields were performed using usually present in infancy or early childhood with the Humphrey Field Analyser II (Carl Zeiss Meditec reduced central vision, with or without . AG, Oberkochen, Germany). Nyctalopia is a prominent feature and some patients also demonstrate photoattraction.4 Visual Molecular biology fi To cite: Hull S, Holder GE, acuity (VA), although signi cantly reduced in child- Bidirectional Sanger sequencing of all exons and Robson AG, et al. Br J hood is usually sufficient to enable a sighted educa- intron–exon boundaries of RPE65 was performed Ophthalmol tion.45The fundus appearance is usually normal in on two probands (1 and 2) with some features of – 2016;100:1499 1505. infancy but small subretinal white dots may appear retinal dystrophy due to mutations in RPE65, but

Hull S, et al. Br J Ophthalmol 2016;100:1499–1505. doi:10.1136/bjophthalmol-2015-308019 1499 Downloaded from http://bjo.bmj.com/ on November 15, 2016 - Published by group.bmj.com Clinical science

better VA than expected. Subsequent to the discovery of a puta- (figure 1). There was a generalised low level of FAF with irregu- tive hypomorphic allele (p.Arg515Trp) in patient 2 and the lar almost radial bands of relatively increased autofluorescence finding of the same allele in a previous report, a panel of 190 in the macula which did not correspond to the disrupted ISe unrelated probands with recessive RCD was Sanger sequenced band on OCT. for exon 14 of RPE65.16 This yielded one further proband Patient 2 presented with mild nyctalopia and photophilia (patient 3), heterozygous for this mutation. Sanger sequencing of before the age of 5 years with no nystagmus and no deterioration all exons and intron–exon boundaries was undertaken to deter- in symptoms over time. When reviewed, age 18 years, VAwas 0.0 mine a second allele. Finally, Sanger sequencing of RPE65 was logMAR in each eye with low myopic correction. Binocular performed in patient 4, with a retinal appearance similar to Esterman visual fields were full at age 17 years except for bilateral fundus albipunctatus, following a report of this phenotypic extreme outside of 70° eccentricity (figure 2). ERG appearance being associated with mutations in RPE65; mutations demonstrated undetectable rod responses but small cone in both RLBP1 and RDH5 had already been excluded.21 responses in keeping with a RCD. PERG was undetectable indi- Genomic DNA was isolated from peripheral blood lymphocytes cating severe macular involvement (figure 3A). Fundus examin- using the Puregene kit (Gentra Puregene Blood Extraction Kit, ation revealed mild midperipheral RPE hypopigmentation and QIAGEN, Manchester, UK). DNA was amplified using specific- white dots. Time-domain OCT demonstrated a normal ISe band ally designed primers by PCR and the resulting fragments were throughout the macula. There was a ring of relatively increased sequenced using standard protocols (details available on request). autofluorescence in the macula on FAF imaging with the back- Mutation nomenclature was assigned in accordance with ground signal otherwise markedly reduced. This ring did not cor- GenBank Accession number NM_000329.2 with nucleotide respond to any ISe band abnormalities on OCTand as such likely position 1 corresponding to the A of the ATG initiation codon. represents relative preservation of normal autofluorescence Variants were identified as novel if not previously reported in rather than abnormally increased autofluorescence. the literature and if absent from dbSNP available at http://www. The third patient also described nyctalopia at a young age ncbi.nlm.nih.gov/projects/SNP/, NHLBI GO Exome Sequencing with reported good VA into his 20 s and onset of central blur Project available at http://evs.gs.washington.edu/EVS/, 1000 from his late 20s only. VA at age 30 was R 0.6 logMAR and L genomes project available at http://www.1000genomes.org/ and 1.0 logMAR but at last review age 44 was R hand movements, the Exome Aggregation Consortium (ExAC) available at http:// L counting fingers with irregular nystagmoid-like movements. exac.broadinstitute.org, all accessed on 18 December 2015. The ERG at age 3 years was reported to demonstrate a severe rod– likely pathogenicity of missense variants was assessed using the cone dysfunction (performed elsewhere). On fundus examin- predictive algorithms of SIFT available at http://sift.jcvi.org and ation, there was disc pallor, attenuated vessels, midperipheral PolyPhen-2 available at http://genetics.bwh.harvard.edu/pph2 intraretinal pigment migration and RPE atrophy of both macula and the conservation of the residues throughout the species ana- and midperiphery. There was loss of the ISe band and outer lysed using Clustal Omega accessed at http://www.ebi.ac.uk/ retina on OCT particularly centrally and temporally. FAF Tools/msa/clustalo/ with protein sequences identified from imaging demonstrated loss of autofluorescence parafoveally and Ensembl accessed at http://www.ensembl.org. Protein modelling throughout the peripheral retina with a ring of preserved auto- of the crystalline structure of RPE65 was performed using fluorescence in the macula. Visual Molecular Dynamics (accessed at http://www.ks.uiuc.edu/ Patient 4 presented with nyctalopia at age 7 years but no nys- Research/vmd/) with Protein Data Bank number 4F3A (http:// tagmus. VA was R 0.0 logMAR and L 0.1 logMAR at last review www.pdb.org). age 26 years with deterioration of nyctalopia and colour vision. Binocular Esterman visual field at age 18 years was full (figure 2). RESULTS Uniocular, Humphrey 30-2 visual fields at age 26 years demon- Four patients from four families were investigated. Clinical data strated paracentral scotoma in both with MD −7.65 DB are summarised in table 1 with retinal imaging in figure 1. right and −6.80 DB left. Electrophysiology age 21 years demon- Patient 1 presented with mild nyctalopia without nystagmus strated a generalised rod more than cone pattern of abnormality in early childhood; there was no subjective deterioration over with severe rod-system dysfunction (figure 3B). There was some time. At first review at age 13 years, VA was R 0.2 logarithm of recovery of rod-dominated ERGs after prolonged dark adapta- the minimum angle of resolution (logMAR) in each eye. At last tion but they did not normalise. The ERG showed mild worsen- review, VA was R 0.2 logMAR and L 0.3 logMAR at age ing of rod function over 12 years of testing. PERG was normal at 22 years with a highly myopic correction. Binocular Esterman age 15 years with deterioration indicating macular dysfunction visual fields performed at age 18 years were full except for an evident at 21 years. Fundus examination revealed widespread dis- extreme left scotoma outside 70° eccentricity (figure 2). crete white dots both round and ovoid in shape throughout the Uniocular 24-2 Humphrey visual fields at age 22 years demon- midperiphery of both eyes predominantly around the arcades strated central scotoma with a mean deviation (MD) of reminiscent of fundus albipunctatus. The number and distribu- −10.26 DA, decibels on the right, −8.34 DB on the left. ERG at tion of the dots remained stable over 12 years of imaging age 13 years showed generalised retinal dysfunction, with cone although occasional dots were observed to fade in size. On OCT ERGs more affected than rod ERGs (figure 3A). PERG showed the white dots extended from the RPE to the level of the external marked macular involvement. Repeat ERG at age 20 years limiting membrane (ELM) (figure 1). The ISe band was disrupted demonstrated no deterioration. Fundus examination demon- but present throughout the macula. FAF imaging demonstrated strated myopic changes with peripapillary atrophy and visible generalised reduction of autofluorescence with a discrete area of choroidal vasculature. There were a few white dots in the pos- further reduction inferonasal to the in the right eye terior pole and extensive granularity of the retinal mid- only, suggestive of RPE atrophy (55° FAF imaging, not shown). periphery that had a white flecked appearance. OCT imaging The white dots were not hyperautofluorescent. revealed a normal inner segment ellipsoid (ISe) band at the Molecular screening of RPE65 in the first patient identified a macula with parafoveal disruption and temporal retinal thinning homozygous variant in RPE65, c.746A>G (p.Tyr249Cys), pre- in both eyes consequent upon outer nuclear layer thinning dicted to be damaging in silico (table 2). This patient’s

1500 Hull S, et al. Br J Ophthalmol 2016;100:1499–1505. doi:10.1136/bjophthalmol-2015-308019 Downloaded from http://bjo.bmj.com/ on November 15, 2016 - Published by group.bmj.com Clinical science

Table 1 Clinical summary Age at Age at last Latest VA Latest Family Age of last Length electrophysiology, key logMAR refractive (gender) onset review follow-up Fundus findings (Snellen) error, dioptres Other findings

Patient 1 (m) 2 years 22 years 9 years Occasional small white 19 years: cone–rod R 0.2 (6/9.5) R −15.50/ Binocular Esterman dots in posterior pole, dysfunction with marked −3.50×169 L fields well-preserved extensive mid-peripheral macular involvement; no −16.50/ age 18, Humphrey 24-2 RPE fleck granularity deterioration over 7 years −3.75×30 central scotoma age 22 GC16768 L 0.3 (6/12) Patient 2 (m) 3 years 19 years 0 years Mild peripheral RPE 17 years: rod–cone R 0.0 (6/6) R −0.50/ Minimal exophoria with hypopigmentation, dysfunction with severe −3.00×175 good recovery occasional white dot macular involvement GC14577 L 0.0 (6/6) L −0.50/ Binocular Esterman −3.25×175 fields well-preserved age 17 Patient 3 (m) 3 years 35 years 13 years Widespread pigmentary 3 years: severe rod–cone R CF Myopic Central visual loss in change with attenuated dysfunction 20s vessels, pale discs GC130 L 1.0 (6/60) Patient 4 (f) 7 years 26 years 15 years Extensive round/ovoid 26 years: rod–cone R 0.0 (6/6) R −4.50 DS Intermittent L white-yellow dots in dysfunction, partial rod mid-periphery and round recovery after prolonged arcades dark adaptation; mild worsening over 12 years PERG normal at age 15 years, reduced at age 21 years GC15862 L0.1 (6/7.5) L +1.00/−1.25 Ishihara age 22, 17/17 each eye, age 26, R 12/ 17 L 16/17 Binocular Esterman fields full age 18, Humphrey 30-2 age 26 paracentral CF, counting fingers vision; DS, dioptre sphere; RPE, retinal pigment epithelium; VA, visual acuity.

molecular result has been previously reported.22 Segregation phenotype of four patients with RPE65 mutations with good VA was confirmed in the family with an affected brother (unavail- and preserved visual fields into adulthood. able for clinical assessment) also homozygous for this variant Four hypomorphic patients with homozygous or compound and their parents and sisters heterozygous. Two mutations in heterozygous missense mutations in RPE65 have previously been RPE65 were found in the second patient; c.1067dupA reported with nyctalopia from early childhood, but normal VA (p.Asn365Lysfs*9), a novel premature truncating codon (PTC) until 6–24 years.4151618The first with reduced central vision in mutation, and c.1543C>T (p.Arg515Trp) which has been previ- the second decade carried two missense variants, p.Leu22Pro ously reported as a hypomorphic allele.16 This allele was also and p.His68Tyr.18 He had end-stage disease with a barely detect- identified in patient 3 as well as a second mutation, c.11 able photopic ERG and undetectable scotopic ERGs when exam- +5G>A, previously reported as one of the commonest variants ined at age 40 years. The second was still able to read age found in RPE65-related disease.4 Screening of RPE65 in patient 40 years and had two missense variants, p.Tyr79His and p. 4 identified compound heterozygous mutations, c.433G>A (p. Glu95Gln; no further information was given on the retinal Ala145Thr, paternal), not previously associated with disease, appearance or ERG.4 The third homozygous for p.Arg515Trp predicted to be tolerated in silico and found in 15 of 121 334 developed central vision loss at age 24 years and had end-stage alleles on ExAC and c.886dupA (p.Arg296Lysfs*7, maternal) disease with an undetectable ERG when examined at age which has been previously reported.23 54 years.16 The fourth homozygous for p.Pro25Leu had good Analysis of the conservation of missense residues throughout a VA when last reviewed at age 7 years.15 He had blonde fundi, broad range of vertebrate and invertebrate classes was performed residual rod function and relatively well preserved cone function with Clustal Omega (see online supplementary figure S1). The p. on ERG and a low but detectable FAF. Apart from the fourth Ala145Thr and p.Arg515Trp arose in completely conserved resi- report, limited phenotypic data have been published. dues, with the p.Tyr249Cys arising in a non-conserved residue. In contrast, the patients reported herein had detailed pheno- Assessment of the position of mutations on the three- typing of both retinal structure and function. All patients had dimensional crystalline structure of RPE65 did not demonstrate good VA until at least age 19 years, with only mild visual field an observable difference between hypomorphic and non- loss. Three have lengthy follow-up with evidence of a slow but hypomorphic residues (see online supplementary figure S2). definite progression based on VA (patients 1 and 3), colour vision testing (patient 4) and ERG/PERG (patient 4). Patients 1 DISCUSSION and 2 had minimal fundus changes at last review, similar to the Although the majority of patients with mutations in RPE65 have previously reported young patient. In contrast, patient 3 showed severe , this study reports the detailed the typical features of advanced photoreceptor degeneration at

Hull S, et al. Br J Ophthalmol 2016;100:1499–1505. doi:10.1136/bjophthalmol-2015-308019 1501 Downloaded from http://bjo.bmj.com/ on November 15, 2016 - Published by group.bmj.com Clinical science

Figure 1 Retinal imaging: (A) colour fundus photographs, (B) fundus autofluorescence (FAF) imaging, (C) optical coherence tomography. Patient 1, right eye, age 19 years, (A) midperipheral retinal pigment epithelial (RPE) granularity, (B) reduced FAF with patchy radial relatively preserved FAFin the macula, (C) parafoveal disrupted inner segment ellipsoid (ISe) band. Patient 2, right eye, age 18 years, (A) mild midperipheral RPE atrophy and white dots, (B) relatively preserved macula FAF, (C) preserved ISe band. Patient 3, left eye, age 35 years, (A) pale disc, attenuated vessels, midperipheral intraretinal pigment migration and RPE atrophy, macular atrophy (B) residual autofluorescence in macula, (C) loss of outer retina and ISe band. Patient 4, right eye, extensive white dots around arcades, (B) generalised reduced FAF with a further area of more markedly reduced FAF inferonasal to the disc, (C.i) disrupted ISe band, (C.ii) white dots extend from RPE to external limiting membrane.

Figure 2 Visual fields for patients 1, 2 and 4. Binocular Esterman suprathreshold fields and Humphrey uniocular threshold fields: patient 1, well-preserved Esterman field at age 18 years, central scotoma on uniocular Humphrey 24-2 at age 22 years; patient 2 well-preserved Esterman field at age 17 years; patient 4 well-preserved Esterman field at age 18 years, arcuate defects on uniocular Humphrey 30-2 at age 26 years.

1502 Hull S, et al. Br J Ophthalmol 2016;100:1499–1505. doi:10.1136/bjophthalmol-2015-308019 Downloaded from http://bjo.bmj.com/ on November 15, 2016 - Published by group.bmj.com Clinical science

Figure 3 Electrophysiology for patients 1, 2 and 4. (A) Right eye (RE) of patients 1 (age 19 years) and 2 (age 18 years) with normal (N) for comparison. Patient 1 shows cone>rod dysfunction of moderate severity with marked macular involvement: the rod-specific electroretinogram (ERG, DA 0.01) is markedly subnormal with DA 10.0 a-wave moderately subnormal; the photopic ERGs (LA 30 Hz and LA 3.0) are markedly delayed and subnormal with a markedly subnormal pattern ERG (PERG). Patient 2 has undetectable rod function with markedly delayed and subnormal cone responses and an undetectable PERG, in keeping with a rod–cone dystrophy with severe macular involvement. The residual bright flash (DA 10.0) likely represents dark-adapted cone system activity, exposed in the absence of rod system function. (B) RE (overnight dark adaptation, DA) and left eye (LE) standard 20 min DA of patient 4 (age 21 years) with N for comparison (DA 20 min); rod-specific ERG is abnormal but detectable from the RE and undetectable from the LE. Bright flash DA ERGs (DA 10.0) show a subnormal a-wave bilaterally, worse on the left; photopic full-field ERGs are reduced and delayed. PERG from the LE is markedly subnormal but when recorded 6 years earlier had been normal bilaterally. The findings are in keeping with marked retinal dysfunction of both rods and cones with partial recovery of rod function after prolonged dark adaptation. DA, dark-adapted; LA, light-adapted. age 35 years similar to the end-stage disease in two previously minimally disrupted or intact ISe band; in patient 3 with reported patients.15 16 18 OCT imaging in patients 1, 2 and 4 advanced disease the OCT was markedly thinned.9 FAF was demonstrated preserved parafoveal retinal thickness and reduced but detectable in all patients.15

Hull S, et al. Br J Ophthalmol 2016;100:1499–1505. doi:10.1136/bjophthalmol-2015-308019 1503 Downloaded from http://bjo.bmj.com/ on November 15, 2016 - Published by group.bmj.com Clinical science

photopic visual function in childhood. For example, Table 2 Mutations found in four families and first report p.Pro25Leu was found to have 7.75% residual isomerase activity Variant: nucleotide, in vitro, sufficient to maintain good VA in a patient at age Family protein Predicted effect First reported 4.5 years.15 In contrast, p.His68Tyr, associated with a severe 1 c.746A>G; p.Tyr249Cys Damaging – Sift Henderson et al22 phenotype, had <2% activity. In compound heterozygous score 0, Polyphen2 (same family) patients with one hypomorphic allele and one severe mutant score 1.0 allele, the overall residual activity appears sufficient to regener- 2 c.1067dupA; p. PTC Novel ate physiologically significant levels of chromophore. Further in Asn356Lysfs*9 vitro assessment would determine the precise impact on isomer- c.1543C>T; p.Arg515Trp Damaging – Sift Kondo et al16 ase activity for the presumed hypomorphic alleles reported in score 0, Polyphen2 our subjects. score 1.0 It appears from the study of patients with hypomorphic 3 c.11+5G>A Splice site Gu et al3 alleles that low levels of isomerase activity are sufficient to main- c.1543C>T; p.Arg515Trp Damaging – Sift Kondo et al16 score 0, Polyphen2 tain good foveal cone function in adulthood despite ERG evi- score 1.0 dence of poor rod and cone function and marked macular 15 4 c.433G>A; p.Ala145Thr Tolerated Sift 0.13, rs767528365 dysfunction on PERG. It is therefore surprising that improve- Polyphen2 0.41 ExAC database 15 ment in VA in RPE65 gene replacement therapy trials has been in 121,334 alleles limited despite good rescue of rod and extrafoveal cone func- et al23 – c.886dupA; p. PTC Coppieters tion.12 14 In the patients we describe, low level of RPE65 activ- Arg296Lysfs*7 ity from birth is associated with normal VA but markedly PTC, premature truncating codon; ExAC, Exome Aggregation Consortium. reduced rod and cone function. This suggests that the efficiency of transduction in human gene replacement trials may not be the limiting factor in improving VA post-treatment, but rather Most patients with RPE65 deficiency have undetectable rod that gene replacement therapy should be given very early in function on ERG but may have residual cone function until late childhood for optimal recovery of VA.12 in the disease.10 Patient 2 had absent rod function but patients 1 and 4 had residual rod function in keeping with milder disease Contributors SH, ARW and ATM designed the study; SH, RM and ARW acquired with similarities to the previously reported 7 year old patient.15 data; all authors analysed data and critically reviewed the manuscript; all authors approved the final manuscript. Unusually, patient 1 demonstrated relatively greater cone than rod dysfunction on ERG examination with no evidence of Funding The National Institute for Health Research (UK) and Biomedical Research Centre at Moorfields Eye Hospital and the UCL Institute of Ophthalmology deterioration between ages 12 and 19. (BRC2_003), Fight For Sight (1318 and 1801), Moorfields Eye Hospital Special Patient 4 had widespread white dots that extended from RPE Trustees (ST1109B). Michel Michaelides is supported by an FFB Career Development to ELM. Fundus albipunctatus due to compound heterozygous Award. RPE65 mutations (IVS1+5G>A and p.Ile115Thr) was previ- Competing interests ATM is the Chairman of Data safety and on the ously reported in a patient who had deterioration of VA by age Management Board of gene therapy trials for retinal disease sponsored by Sanofi 18.21 Electroretinography was similar to that found in RDH5 and Oxford Biomedica and has served on an Advisory Board for Sucampo Pharmaceuticals and Sanofi. He has also participated in a clinical trial of oral mutations with normalisation of rod-system ERGs after pro- retinoid therapy for RPE65 deficiency sponsored by QLT. longed dark adaptation. The patient in the present series has a Ethics approval NRES Committee London – Camden amp; Islington, Research similar retinal appearance but only partial recovery of rod func- Management Committee, Moorfields. tion following extended dark adaptation and excellent VA at age Provenance and peer review Not commissioned; externally peer reviewed. 26 years. The dots in the previously reported patient were demonstrated on OCT to extend throughout the RPE up to the level of the ELM, similar to the patient in this report and to a REFERENCES fi RPE65- 1 Moiseyev G, Chen Y, Takahashi Y, et al. RPE65 is the isomerohydrolase in the previous report of OCT ndings in an related severe – 6 retinoid visual cycle. Proc Natl Acad Sci USA 2005;102:12413 18. early onset retinal dystrophy patient. 2 Marlhens F, Bareil C, Griffoin JM, et al. Mutations in RPE65 cause Leber’s Molecular analysis in patient 1 identified a homozygous congenital amaurosis. Nat Genet 1997;17:139–41. p.Tyr249Cys variant, predicted to be pathogenic in silico. 3 Gu SM, Thompson DA, Srikumari CR, et al. Mutations in RPE65 cause autosomal Patients 2 and 3 carried two variants including the p.Arg515Trp recessive childhood-onset severe retinal dystrophy. Nat Genet 1997;17:194–7. allele that has been previously reported in a similarly mildly 4 Thompson DA, Gyürüs P, Fleischer LL, et al. Genetics and phenotypes of RPE65 16 mutations in inherited retinal degeneration. Invest Ophthalmol Vis Sci affected patient. Patient 4 had a missense change, 2000;41:4293–9. p.Ala145Thr, tolerated in silico and not previously reported in 5 Lorenz B, Gyürüs P, Preising M, et al. Early-onset severe rod-cone dystrophy in disease, but arising in a highly conserved residue. This was com- young children with RPE65 mutations. Invest Ophthalmol Vis Sci 2000;41:2735–42. bined with a PTC mutation, previously reported in a patient 6 Weleber RG, Michaelides M, Trzupek KM, et al. The phenotype of Severe Early 23 Childhood Onset Retinal Dystrophy (SECORD) from mutation of RPE65 and with LCA and assumed to be null. The analysis of mutations differentiation from Leber congenital amaurosis. Invest Ophthalmol Vis Sci in this series of patients would indicate that p.Tyr249Cys, 2011;52:292–302. p.Arg515Trp and p.Ala145Thr are all hypomorphic alleles. 7 Galvin JA, Fishman GA, Stone EM, et al. Evaluation of genotype-phenotype Investigation of the location of hypomorphic residues in the associations in leber congenital amaurosis. Retina 2005;25:919–29. fl crystalline RPE65 structure revealed no apparent difference 8 Lorenz B, Wabbels B, Wegscheider E, et al. Lack of fundus auto uorescence to 488 nanometers from childhood on in patients with early-onset severe retinal dystrophy when compared with non-hypomorphic residues. associated with mutations in RPE65. Ophthalmology 2004;111:1585–94. A previously reported in vitro assay system using purified 9 Jacobson SG, Cideciyan AV, Aleman TS, et al. Photoreceptor layer topography in RPE65 confirmed its isomerase function in the visual cycle and children with leber congenital amaurosis caused by RPE65 mutations. Invest 24 Ophthalmol Vis Sci 2008;49:4573–7. permitted quantitative analysis of enzymatic activity. Some fi RPE65 10 Jacobson SG, Aleman TS, Cideciyan AV, et al.De ning the residual vision in leber hypomorphic alleles have been shown in vitro to congenital amaurosis caused by RPE65 mutations. Invest Ophthalmol Vis Sci produce RPE65 protein with sufficient residual activity for good 2009;50:2368–75.

1504 Hull S, et al. Br J Ophthalmol 2016;100:1499–1505. doi:10.1136/bjophthalmol-2015-308019 Downloaded from http://bjo.bmj.com/ on November 15, 2016 - Published by group.bmj.com Clinical science

11 Kaylor JJ, Cook JD, Makshanoff J, et al. Identification of the 11-cis-specific 18 Marlhens F, Griffoin JM, Bareil C, et al. Autosomal recessive retinal dystrophy retinyl-ester synthase in retinal Müller cells as multifunctional O-acyltransferase associated with two novel mutations in the RPE65 gene. Eur J Hum Genet (MFAT). Proc Natl Acad Sci USA 2014;111:7302–7. 1998;6:527–31. 12 Cideciyan AV, Jacobson SG, Beltran WA, et al. Human retinal gene therapy for Leber 19 McCulloch DL, Marmor MF, Brigell MG, et al. ISCEV standard for full-field clinical congenital amaurosis shows advancing retinal degeneration despite enduring visual electroretinography (2015 update). Doc Ophthalmol 2015;130:1–12. improvement. Proc Natl Acad Sci USA 2013;110:E517–25. 20 Bach M, Brigell MG, Hawlina M, et al. ISCEV standard for clinical 13 Testa F, Maguire AM, Rossi S, et al. Three-year follow-up after unilateral subretinal pattern electroretinography (PERG): 2012 update. Doc Ophthalmol delivery of adeno-associated virus in patients with Leber congenital Amaurosis type 2013;126:1–7. 2. Ophthalmology 2013;120:1283–91. 21 Schatz P, Preising M, Lorenz B, et al. Fundus albipunctatus associated 14 Bainbridge JW, Mehat MS, Sundaram V, et al. Long-term effect of gene therapy on with compound heterozygous mutations in RPE65. Ophthalmology Leber’s congenital amaurosis. N Engl J Med 2015;372:1887–97. 2011;118:888–94. 15 Lorenz B, Poliakov E, Schambeck M, et al. A comprehensive clinical and biochemical 22 Henderson RH, Waseem N, Searle R, et al. An assessment of the apex microarray functional study of a novel RPE65 hypomorphic mutation. Invest Ophthalmol Vis Sci technology in genotyping patients with Leber congenital amaurosis and early-onset 2008;49:5235–42. severe retinal dystrophy. Invest Ophthalmol Vis Sci 2007;48:5684–9. 16 Kondo H, Qin M, Mizota A, et al. A homozygosity-based search for mutations in 23 Coppieters F, Casteels I, Meire F, et al. Genetic screening of LCA in Belgium: patients with autosomal recessive pigmentosa, using microsatellite markers. predominance of CEP290 and identification of potential modifier alleles in AHI1 of Invest Ophthalmol Vis Sci 2004;45:4433–9. CEP290-related phenotypes. Hum Mutat 2010;31:E1709–66. 17 Bowne SJ, Humphries MM, Sullivan LS, et al. A dominant mutation in RPE65 24 Nikolaeva O, Takahashi Y, Moiseyev G, et al. Purified RPE65 shows identified by whole-exome sequencing causes with choroidal isomerohydrolase activity after reassociation with a phospholipid membrane. involvement. Eur J Hum Genet 2011;19:1074–81. FEBS J 2009;276:3020–30.

Hull S, et al. Br J Ophthalmol 2016;100:1499–1505. doi:10.1136/bjophthalmol-2015-308019 1505 Downloaded from http://bjo.bmj.com/ on November 15, 2016 - Published by group.bmj.com

Preserved visual function in retinal dystrophy due to hypomorphic RPE65 mutations

Sarah Hull, Graham E Holder, Anthony G Robson, Rajarshi Mukherjee, Michel Michaelides, Andrew R Webster and Anthony T Moore

Br J Ophthalmol 2016 100: 1499-1505 originally published online February 23, 2016 doi: 10.1136/bjophthalmol-2015-308019

Updated information and services can be found at: http://bjo.bmj.com/content/100/11/1499

These include: References This article cites 24 articles, 11 of which you can access for free at: http://bjo.bmj.com/content/100/11/1499#BIBL Email alerting Receive free email alerts when new articles cite this article. Sign up in the service box at the top right corner of the online article.

Topic Articles on similar topics can be found in the following collections Collections Epidemiology (1066) Vision (623)

Notes

To request permissions go to: http://group.bmj.com/group/rights-licensing/permissions

To order reprints go to: http://journals.bmj.com/cgi/reprintform

To subscribe to BMJ go to: http://group.bmj.com/subscribe/