ARVO 2015 Annual Meeting Abstracts 317 Genetics of retinal dystrophies and RP Tuesday, May 05, 2015 8:30 AM–10:15 AM Exhibit Hall Poster Session Program #/Board # Range: 2857–2901/C0113–C0157 Organizing Section: Genetics Group Contributing Section(s): Biochemistry/Molecular Biology, Low Vision, Retinal Cell Biology, Retina Program Number: 2857 Poster Board Number: C0113 Presentation Time: 8:30 AM–10:15 AM Digenic inheritance of heterozygous RP1L1 and C2orf71 variants in syndromic retinitis pigmentosa Frans Cremers1, 2, Yangfan P. Liu3, Danielle G. Bosch1, 5, Anna M. Siemiatkowska1, 2, Nanna D. Rendtorff4, Christian Gilissen1, 2, F. N. Boonstra5, Claes Moller6, Lisbeth Tranebjærg4, Nicholas Katsanis3. 1 Human Genetics, Radboud University Medical Center, Nijmegen, Netherlands; 2Radboud Institute for Molecular Life Sciences, Radboud University, Nijmegen, Netherlands; 3Duke University School of Medicine, Durham, NC; 4University of Copenhagen, Copenhagen, Denmark; 5Institute for the Visually Impaired, Zeist, Netherlands; 6Faculty of Medicine and Health, Orebro, Sweden. Purpose: To identify the genetic cause for a syndrome identified in an isolated case, which consisted of retinitis pigmentosa (RP), hearing loss, ataxia and cerebellar atrophy. Methods: The presence of copy number variants (CNVs) were investigated using a genome-wide CGH array and by MLPA analysis of OPA1 and WFS1. Candidate disease genes (CEP290, GJB2, OPA1, WFS1, ZCD2) and mitochondrial DNA variants were analysed by Sanger sequencing. Allele-specific primer extension analysis was performed using microarrays containing variants implicated in Leber congenital amaurosis, autosomal recessive and dominant RP, and Usher syndrome. The exome of the proband was enriched using the SureSelect Human All Exon v4 Kit (50 Mb), and sequenced on a 5500XL platform. Morpholino oligonucleotides (MOs) and CRISPR gRNAs were designed for zebrafish genes rp1l1 and C2orf71like for knockdown and knockout, respectively. MOs or CRISPR gRNAs plus Cas9 RNA were microinjected into the yolk of zebrafish embryos at 1-8 cell stage, and convergent extension phenotype was examined at 10 somite stage. At 5 days post fertilization, lateral images of zebrafish larvae were taken to measure eye size. Larvae were then analysed by histoimmunostaining. Results: No CNVs, mutations in known disease genes or mitochondrial DNA defects were found that could account for the phenotype under an autosomal recessive paradigm. However, exome sequencing revealed a complex heterozygous proteintruncating mutation in RP1L1, p.[(K111Qfs*27; Q2373*)], and a heterozygous nonsense mutation in C2orf71, p.(S512*) on the other allele. Mutations in both genes have been implicated previously in autosomal recessive nonsyndromic RP, raising the possibility of a digenic model in this family. Functional testing for two key phenotypes of the patient in zebrafish showed that the combinatorial suppression of rp1l1 and c2orf71l induced discrete pathology in terms of reduction of eye size with concomitant loss of rhodopsin in the photoreceptors, and disorganization of the cerebellum. Conclusions: We propose that the combination of heterozygous loss-of-function mutations in these genes drives syndromic retinitis pigmentosa, likely through the genetic interaction of at least two loci, whereas haploinsuffiency of each is insufficient to induce overt pathology. Commercial Relationships: Frans Cremers, None; Yangfan P. Liu, None; Danielle G. Bosch, None; Anna M. Siemiatkowska, None; Nanna D. Rendtorff, None; Christian Gilissen, None; F. N.

Boonstra, None; Claes Moller, None; Lisbeth Tranebjærg, None; Nicholas Katsanis, None Support: FP7-PEOPLE-2012-ITN programme EyeTN, agreement 317472, the Macula Vision Research Foundation Program Number: 2858 Poster Board Number: C0114 Presentation Time: 8:30 AM–10:15 AM The mutation spectrum of a large Brazilian inherited retinal disease cohort Fernanda B. Porto1, 2, Zachry Soens3, Shirley A. Sampaio1, Igor M. Maia4, Millena C. Sousa4, Yumei Li3, Shan Xu3, Katharina Schulze3, Renata T. Simoes4, Rui Chen3. 1INRET - Clínica e Centro de Pesquisa, Belo Horizonte, Brazil; 2Departamento de Retina e Vítreo, Centro Oftalmológico de Minas Gerais, Belo Horizonte, Brazil; 3Human Genome Sequencing Center Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX; 4Instituto de Ensino e Pesquisa da Santa Casa de Belo Horizonte, Belo Horizonte, Brazil. Purpose: The purpose of this study was to examine the mutation spectrum of a largely unstudied ethnicity which comprises the world’s fifth most populous nation in order to assign a molecular diagnosis to patients, enable access to clinical trials and emerging treatments, and to report the identified pathogenic variants to the retinal disease community to advance the study of retinal diseases. Methods: Brazilian consenting patients were recruited starting in July 2012. The study protocol adhered to the tenets of the Declaration of Helsinki and was approved by the local ethics committee. 1492 patients are available in this cohort with various diseases including Retinitis Pigmentosa (RP), Stargardt (STGD)/STGD-like disease, Usher syndrome, and Leber Congenital Amaurosis (LCA). Genomic DNA was extracted from patients’ blood samples and genotyped by capture sequencing of 224 known retinal disease genes using next-generation sequencing (NGS). Captured DNA was sequenced on the Illumina HiSeq platform and NGS data was processed by an in-house bioinformatics pipeline leading to annotated variant calls. Patients with negative results from our panel sequencing were further analyzed by whole exome sequencing. The results of the first 300 patients are being reported herein. Results: A molecular diagnosis was able to be assigned in 80% of LCA, 60% of RP, 35% of STGD/STGD-like, and 80% of Usher syndrome. Approximately 70% of causative variants identified in this study were novel when compared to variants reported to be pathogenic in the Human Gene Mutation Database. Conclusions: The solving rates obtained for each disease cohort are similar to published cohorts of other ethnicities found in literature, however the distribution of which genes are affected differs. In Caucasian LCA patients CEP290 is the most widely affected gene, while in Brazilians CRB1 accounts for the majority of disease. In Brazilian RP patients RHO, EYS, and PRPH2 are the most frequently mutated genes, while in Chinese and Caucasian RP patients USH2A and ABCA4 are two of the most frequently mutated. Discovering the genetic basis of retinal disease in unstudied ethnicities remains an important method to expand our knowledge about the different causes of retinal disease which can help lead to treatments. Commercial Relationships: Fernanda B. Porto, None; Zachry Soens, None; Shirley A. Sampaio, None; Igor M. Maia, None; Millena C. Sousa, None; Yumei Li, None; Shan Xu, None; Katharina Schulze, None; Renata T. Simoes, None; Rui Chen, None

©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected].

ARVO 2015 Annual Meeting Abstracts Program Number: 2859 Poster Board Number: C0115 Presentation Time: 8:30 AM–10:15 AM Identification of the causal variants for different inherited retinal phenotypes in a complex consanguineous family from Pakistan Maleeha Maria2, 1, Maleeha Azam2, 1, Syeda H. Ali2, 3, Frans Cremers1, 4 , Raheel Qamar2, 5, Muhammad I. Khan1, 2, Lonneke Haer-Wigman1, 4 1 . Human Genetics, Radboud University Medical Center, Nijmegen, Netherlands; 2Biosciences, COMSATS Institute of Information Technology, Islamabad, Pakistan; 3Institute of Pure and Applied Biology, Bahauddin Zakariya University, Multan, Pakistan; 4Radboud Institute for Molecular Life sciences, Radboud University, Nijmegen, Netherlands; 5Al-Nafees Medical College & Hospital, Isra University, Islamabad, Pakistan. Purpose: A large consanguineous Pakistani family with multiple loops in which the affected individuals suffered from Leber congenital amaurosis (LCA), autosomal recessive retinitis pigmentosa (arRP), or cone dysfunction (CD) was analyzed for the identification of causal genetic defects in this family by using homozygosity mapping and whole exome sequencing (WES). Methods: We performed homozygosity mapping in two LCA siblings using the Illumina 700k single nucleotide polymorphism microarray. Six retinal disease genes (IQCB1, KLHL7, MERTK, NPHP3, RBP3, and RHO) residing in the shared homozygous regions larger than 2 Mb were sequenced. One LCA patient was subsequently analyzed using WES and variants with a PhyloP >2.7 and dbSNP frequency 10 genotypes each were located in nucleotidebinding domains (78%). Outstanding mutation hotspots were mapped to the TM (G863A, with 64 genotypes), the NBD1 domain (G1961E, 111; A1038V, 48; R1108C, 22; N965S, 13; and E1087K, 10 genotypes), and the NBD2 domain (L2027F, 31; R2107H, 24; R2030W, 17; and V2050L, 11 genotypes). Molecular modeling demonstrated that hotspots located at domain surface were severe and perturbed the ATP-hydrolysis. Mutation G863A affected protein flippase activity. Conclusions: The large-scale mutational analysis and predictions of mutation severities from atomic level could be useful for the functional annotation of genetic variants from next-generation sequencing data and establishing the genotype-to-phenotype relationships in genetic disease. Commercial Relationships: Yuri V. Sergeev, None; Katherine Pogrebniak, None; Kaoru Fujinami, None; Benedetto Falsini, None; Wadih M. Zein, None; Kerry E. Goetz, None; Thiran Jayasundera, None; Michel Michaelides, None; Brian P. Brooks, None; Paul A. Sieving, None Support: NIH IRP ZIA EY000476-06

©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected].

ARVO 2015 Annual Meeting Abstracts Program Number: 2861 Poster Board Number: C0117 Presentation Time: 8:30 AM–10:15 AM The clinical course of Stargardt disease in a French cohort of patients Saddek Mohand-Said2, 1, Catherine Brun-Strang3, Ronald BUGGAGE4, Konstantinos Aliferis1, Isabelle S. Audo2, 1, Caroline Amand3, Ieva Sliesoraityte1, Alain Stemart5, Jose A. Sahel2, 1. 1CHNO Quinze-Vingts / CIC Inserm, Paris, France; 2UPMC Paris VI, Paris, France; 3Sanofi R&D HEOR, Paris, France; 4Sanofi R&D, Division medical officer, Ophthalmology Unit, Paris, France; 5Aixial Pharma, Levallois-Perret, France. Purpose: Stargardt disease (SD), the most common hereditary macular dystrophy, is a rare disease caused by mutations in the ABCA4 gene. In the literature, only scarce epidemiologic data are available to document on the natural course of SD. The objective of this study was to measure the progression of the disease using a variety of clinical parameters and to better understand the disease burden as derived from quality of life (QoL) scales. Methods: Single center retrospective observational clinical study on an existing cohort of 148 French patients with SD followed in the Centre Hospitalier National d’Ophtalmologie (CHNO) des Quinze-Vingts. 26 patients genotyped with at least one pathogenic mutant ABCA4 allele on each chromosome and followed with at least two recorded visits were included. Clinical data including best corrected visual acuity changes (BCVA), fundus examination, visual field (Goldmann kinetic perimetry and Static periletry [Octopus]), optical coherence tomography (OCT), fundus autofluorescence (FAF), and electroretinography (Full-field ERG and Multifocal-ERG) were collected during an initial visit and follow-up visit(s) (FUV1 / FUV2). Quality of life (QoL) was evaluated using 2 questionnaires: Hospital Anxiety and Depression scale (HADS) and Visual Function Questionnaire 25 (NEI-VFQ-25). Results: Preliminary descriptive data (57% Male) showed that the mean age of the cohort was 34.2 years (median 32.5) at baseline visit. The mean age at symptoms onset was 17.2 (10 to 29 years, median 17.5). At inclusion, 54% patients were categorized as SD type 1, 31% as type 2 and 15% as type 3, according to the classification based on the ERG phenotype (Lois et al, Arch Ophthalmol., 2001. At FU V1, (average of 2.7 years after the initial visit), 42% patients were type 1, 33% type 2 and 25% type 3. Absolute mean change in BCVA from inclusion to first visit of follow-up was 1.9 ± 4.2 ETDRS letters. No significant changes were noted on the QoL scales and on the other clinical parameters studied during the follow-up period. Conclusions: On review of these preliminary data no evidence of significant clinical disease progression or QoL change was observed in our cohort during the follow-up interval. This may be due to the advanced stage of the disease at the time of initial visit. It is expected that more evidence of progression may have been seen if the cohort had been observed from earlier stage of the disease. Commercial Relationships: Saddek Mohand-Said, None; Catherine Brun-Strang, None; Ronald BUGGAGE, None; Konstantinos Aliferis, None; Isabelle S. Audo, None; Caroline Amand, None; Ieva Sliesoraityte, None; Alain Stemart, None; Jose A. Sahel, None Support: FFB center grant C-CMM-0907-0428-INSERM04, C-CL0912-0600-INSERM01 Clinical Trial: EUDRACT 2006-A00347-44

Program Number: 2862 Poster Board Number: C0118 Presentation Time: 8:30 AM–10:15 AM ABCA4 variants in Stargardt disease: Preliminary results from the ProgStar study Samantha Bomotti1, 2, Rupert W. Strauss3, 2, Hendrik P. Scholl2, Robert Wojciechowski1, 2. 1Epidemiology, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD; 2Johns Hopkins Wilmer Eye Institute, Baltimore, MD; 3Department of Ophthalmology, Medical University Graz, Graz, Austria. Purpose: Stargardt disease type 1 (OMIM 248200) is the most common juvenile macular dystrophy. It follows an autosomal recessive mode of inheritance. There is currently no cure and Stargardt remains difficult to diagnose due to its high allelic and phenotypic heterogeneity. We are compiling phenotypic data and genetic variant information at the ABCA4 gene in Stargardt patients at nine clinical centers across the United States and Europe. The data will be used to establish genotype-phenotype relationships for ABCA4 mutations, and to identify the effects of variants of unknown clinical significance. Methods: To date, genetic and clinical data have been collected from 244 Stargardt patients. A variety of biological assays have been employed to identify the ABCA4 variants, depending on the technologies in use at the time of diagnosis (from 2008 or earlier to present). We report on the distribution of ABCA4 variants in this selected Stargardt population. These preliminary analyses are being conducted as the ProgStar study nears enrollment completion, at which point complete data will be available for in-depth analysis. Results: Among the 244 Stargardt individuals for whom we have genotype information thus far, the majority (86.9%) were heterozygous for ABCA4 mutations. In 26 cases (10.7%), only one ABCA4 mutation was identified, suggesting incomplete coverage of the functional regions of ABCA4 or locus heterogeneity. The most common variant was c.5882G>A, found in 67 (27.5%) participants. Three (4.5%) of these individuals were homozygous for this variant. The second most common mutation, c.2588G>C, was observed in one copy in 28 (11.5%) individuals. These variants are among the most commonly found in previously published data. Thirty-three individuals (13.5%) had three putatively pathogenic ABCA4 variants and three (1.2%) had four variants. Conclusions: Initial mutation analyses in the ProgStar study confirm the high allelic heterogeneity of Stargardt disease. These data will shed new light on variant effects on the progression of Stargardt disease. It should be noted that testing protocols varied considerably across testing laboratories that contributed data to ProgStar. Most assays were based on mutations known at the time of testing, and favored screening of more common mutations. High-throughput sequencing technologies will allow for an unbiased ascertainment of pathogenic ABCA4 mutations in Stargardt disease. Commercial Relationships: Samantha Bomotti, None; Rupert W. Strauss, None; Hendrik P. Scholl, QLT Inc. (C), QLT Inc. (F), Sanofi-Fovea Pharmaceuticals (C), Vision Medicines, Inc. (C); Robert Wojciechowski, None

©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected].

ARVO 2015 Annual Meeting Abstracts Support: Samantha Bomotti is supported by NEI T32 grant number: 1T32EI022303, the Eye and Vision Genomics Training Program. Dr. Robert Wojciechowski is supported by NEI grant number: K08EI022943. Dr. Rupert Strauss is supported by Austrian Scientific Fonds Erwin-Schroedinger Stipend Project number J 3383-B23. This entire ProgStar project, and through this Dr. Hendrik Scholl, is supported by the Foundation Fighting Blindness Clinical Research Institute (FFB CRI) and Dr. Scholl is also supported by a grant to FFB CRI by the U.S. Department of Defense USAMRMC TATRC, Fort Meade, Maryland (grant numbers W81-XWH-07-1-0720 and W81XWH-09-2-0189); The Shulsky Foundation, New York, NY; Ocular Albinism Research Fund (Clark Enterprises Inc.); Unrestricted grant to the Wilmer Eye Institute from Research to Prevent Blindness; Baylor-Johns Hopkins Center for Mendelian Genetics (National Human Genome Research Institute, NHGRI/NIH; Identification number: 1U54HG006542-01). H.P.N.S. is the Dr. Frieda Derdeyn Bambas Professor of Ophthalmology. Program Number: 2863 Poster Board Number: C0119 Presentation Time: 8:30 AM–10:15 AM Whole-exome sequencing in patients with STGD (ABCA4)-like phenotypes Yajing Xie1, Winston Lee1, Stephen H. Tsang1, Gerald A. Fishman2, Frederick T. Collison2, Rosa Riveiro-Alvarez3, Carmen Ayuso3, Tomasz Gambin4, James R. Lupski4, Rando Allikmets1. 1Columbia University, New York, NY; 2The Pangere Center for Hereditary Retinal Diseases, The Chicago Lighthouse for People Who Are Blind or Visually Impaired, Chicago, IL; 3Department of Genetics, Instituto de Investigacion Sanitaria-University Hospital Fundacion Jimenez Diaz (IIS-FJD), Madrid, Spain; 4Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX. Purpose: To determine the genetic cause of the disease in patients where clinical diagnosis was compatible with ABCA4-associated phenotypes (Stargardt disease, cone-rod dystrophy, bull’s eye maculopathy and atypical arRP), but where no two disease-associated ABCA4 mutations were identified. Methods: Detailed medical history and ophthalmic examination, including fundus photography, autofluorescence imaging, spectraldomain optical coherence tomography (SD-OCT) and, in some cases, ISCEV-standardized electroretinography, were performed on all patients. The ABCA4 gene was screened in all affected individuals with negative results. Subsequently, affected and available unaffected members from each family and sporadic cases were subjected to whole exome sequencing (WES) on Illumina platform. Possibly disease-associated variants were determined by filtering based on minor allele frequency and predicted pathogenicity. Variants were verified by Sanger sequencing followed by segregation analysis. Results: Ophthalmological examination identified 40 families and 26 sporadic cases with phenotypes compatible with STGD (42), CRD (6), arRP (3), and with BEM (15) all of whom harbored no or one disease-associated ABCA4 mutations after initial analysis. In total, 141 individuals were subjected to WES analysis. The definite causal gene was identified in 22/40 families. Analysis of 15/40 families resulted in several candidate genes, while no plausible candidate was found in 3 families. Of the 26 sporadic cases, 12 were solved, 9 had multiple plausible candidate genes, and 5 were impossible to solve. The causal genes for the solved cases belong to the following categories: 1) phenotypic expansion in known retinal disease genes (11 cases; CRB1, CRX, etc.); 2) revised clinical diagnosis or ambiguous phenotypes (12 cases; RS1, PRPH2, RP1L1, etc.); 3) new non-coding or re-assessed mutations in ABCA4 (7 cases); 4) new genes (RDH11, RAB28) in 3 families; 5) unusual clinical outcome, MMACHC, in one case.

Conclusions: Whole-exome sequencing identified the causal gene in >50% of cases with phenotypes compatible with ABCA4-associated diseases. The genetic causes of ABCA4-like phenotypes range from new genes to unusual clinical manifestations of known genes. Commercial Relationships: Yajing Xie, None; Winston Lee, None; Stephen H. Tsang, None; Gerald A. Fishman, None; Frederick T. Collison, None; Rosa Riveiro-Alvarez, None; Carmen Ayuso, None; Tomasz Gambin, None; James R. Lupski, None; Rando Allikmets, None Support: Supported in part by NIH grants EY021163, EY019861, EY019007, HG006542, and from Research to Prevent Blindness. Program Number: 2864 Poster Board Number: C0120 Presentation Time: 8:30 AM–10:15 AM Molecular analysis of ABCA4 gene in cohort of Chinese patients with Stargardt disease or cone-rod dystrophy Yang Li, Zhe Pan, Feng Jiang, Ke Xu, Xiaohui Zhang, Ning Lu. Beijing Inst of Ophthalmology, Beijing Tongren Hospital, Beijing, China. Purpose: Mutations in the ABCA4 gene can cause autosomal recessive Stargardt disease (STGD), cone or cone-rod dystrophy (CRD), and retinitis pigmentosa. The objective of this study was to find the possible disease causing variants of ABCA4 gene in a cohort of Chinese patients with STGD or CRD and described the correlation between the phenotype and genotype. Methods: A total of 161 probands were recruited for genetic analysis; these included 111 patients diagnosed with STGD and 50 individuals with CRD. All probands underwent ophthalmic examinations including best corrected visual acuity, fundus examination, optical coherence tomography, fundus autofluorescence, and electroretinograms. Genomic DNA was extracted from venous blood of all participants and all coding exons and exon-intron boundaries of the ABCA4 gene were screened for mutations by PCR-based DNA sequencing, followed by analyses for pathogenicity by in silico programs. Restriction fragment length polymorphism analysis or allele specific PCR was used to validate the substitution in all available family members. Results: We found at least two disease-causing alleles in 90 unrelated patients (56%), one disease-causing allele in 23 patients (14%), and no disease-causing allele in 58 affected individuals (30%). In total, 122 disease-causing variants of the ABCA4 gene, including 72 novels, were identified. The identified mutations included 69 (56.7%) missense, 15 (12.3%) nonsense, 21 (17.2%) splicing effect, and 17 (13.9%) indel mutations. The most frequent mutation in this cohort was c.2424C>G p.Y808X, representing 7.2% of all disease alleles. 102 (102/111, 92%) STGD patients were found carrying two or one disease-causing allele of the ABCA4 gene, however, only 11 (11/50, 22%) patients with CRD were identified harboring two or one disease-causing allele. Conclusions: Our results further confirmed that the ABCA4 gene is the most common disease-causing gene for Chinese patients with autosomal recessive STGD. The mutation spectrum of the ABCA4 gene is different from the ones of Caucasian patients. Commercial Relationships: Yang Li, None; Zhe Pan, None; Feng Jiang, None; Ke Xu, None; Xiaohui Zhang, None; Ning Lu, None

©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected].

ARVO 2015 Annual Meeting Abstracts Program Number: 2865 Poster Board Number: C0121 Presentation Time: 8:30 AM–10:15 AM Mutations in the Unfolded Protein Response regulator, ATF6, cause Achromatopsia Susanne Kohl1, Ditta Zobor1, Wei-Chieh Chiang2, Nicole Weisschuh1, Mathias W. Seeliger1, Stanley Chang4, Randal J. Kaufman3, Stephen H. Tsang4, Bernd Wissinger1, Jonathan H. Lin2. 1Centre for Ophthalmology, Inst for Ophthalmic Rsrch Tuebingen, Tuebingen, Germany; 2Department of Pathology, University of California San Diego, La Jolla, CA; 3Center for Neuroscience, Aging, and Stem Cell Research, Sanford Burnham Medical Research Institute, La Jolla, CA; 4Department of Ophthalmology, Columbia University, New York, NY. Purpose: Achromatopsia (ACHM) is an autosomal recessive disorder characterized by color blindness, marked photophobia, nystagmus, and severely reduced visual acuity. In our cohort, disease-causing variants in genes encoding components of the cone phototransduction cascade cause ~75% of cases. Here, we examined patients with ACHM lacking disease-causing variants in the known ACHM genes to identify additional genes associated with this rare disorder. Methods: Genetic analysis included autozygosity mapping, whole exome sequencing, and Sanger-sequencing of ATF6 in over 300 ACHM patients. These studies were complemented by cDNA analysis out of patients’ PaxGene blood to investigate the effect of putative splice site mutations, functional studies to assess the pathogenic effect of a ATF6 missense mutation, as well as functional analysis of the homologous Atf6-/- mouse model. Results: We identified ten independent ACHM families with 18 affected individuals carrying six homozygous and two compoundheterozygous disease-causing variants in the Activating Transcription Factor 6 (ATF6) gene, a key regulator of the Unfolded Protein Response (UPR) and cellular endoplasmic reticulum (ER) homeostasis. The mutations included two missense, three frameshifting indel, and three true splice site mutations – as proven by cDNA analysis. Clinically and on optical coherence tomography the patients had evidence of foveal hypoplasia with an essentially absent foveal pit and a variable degree of disruption of the cone photoreceptor layer at the macula. Functionally, we found that an ACHM-associated ATF6 missense variant leads to attenuated ATF6 transcriptional activity in response to ER stress. Young Atf6-/- mice have normal retinal morphology and function, with both rod and cone dysfunction in older mice. Conclusions: Mutations in ATF6 account for 1% of ACHM cases in our cohort of over 950 independent ACHM families. Inactivating mutations in ATF6 can result in an isolated retinal photoreceptor phenotype despite its ubiquitous expression. This study suggests a crucial and unexpected role of ATF6 in human foveal development and cone photoreceptor function. Commercial Relationships: Susanne Kohl, None; Ditta Zobor, None; Wei-Chieh Chiang, None; Nicole Weisschuh, None; Mathias W. Seeliger, None; Stanley Chang, None; Randal J. Kaufman, None; Stephen H. Tsang, None; Bernd Wissinger, None; Jonathan H. Lin, None Support: BMBF grant 01GM1108A to BW & SK; NIH grants EY001919 & EY020846 to JHL; DK042394, DK088227 & HL052173 to RJK; R01EY018213 to SHT; post-doctoral FFB fellowship to W-CJC.

Program Number: 2866 Poster Board Number: C0122 Presentation Time: 8:30 AM–10:15 AM ARRP microarray and Exome analysis revealed known and novel mutations in Mexican pedigrees Adda L. Villanueva1, 2, Mathieu Langlois4, Ian Mongrain4, Sylvie Provost4, Géraldine Asselin4, Marie-Pierre Dubé4, 5, Keerti Tadimeti3, John Suk3, Pooja Biswas3, Radha Ayyagari3. 1Retina-Genomics, Virtual Eye Care MD, Merida, Mexico; 2Retina-Genomics, Hopital Maisonneuve Rossemont, Montreal, QC, Canada; 3Retina-Genomics, Jacob’s Retina Center, University of California, San Diego, CA; 4 Pharmacogenomics, Montreal Heart Institute and the BeaulieuSaucier Pharmacogenomics Center, Montreal, QC, Canada; 5Faculty of Medicine, Université de Montréal, Montreal, QC, Canada. Purpose: To identify possible causative mutations in four unrelated families with autosomal recessive retinal degeneration. Methods: Clinical analysis included full ophthalmic exam, refraction, Goldman kinetic or Humphrey dynamic perimetry, Amsler test, fundus photography, Ocular coherent tomography, fluorescein angiography and color vision test. Blood samples were collected from affected and unaffected family members and DNA isolated using standard protocol. DNA was analyzed for known mutations using Asper arRP and arLCA array. In addition, exome of one proband was captured using Nimblegen V3, and sequencing was performed on Illumina HiSeq. Reads were mapped against hg19, and variants were identified using GATK. Variants detected in these individuals were analyzed by exomeSuite using an autosomal recessive model. Segregation and ethnicity matched control sample analysis were performed by Dideoxy sequencing. Results: Four families comprising 3-6 in each pedigree with 1-3 affected members were recruited from Yucatan Peninsula, Mexico. Affected members were diagnosed with LCA and cone/rod dystrophy and RP-Usher type. A homozygous missense change p.C759F segregating with disease was found in the USH2A gene in one of the pedigrees. This change was previously described in other patients. In a second pedigree another previously described homozygous change p.P575L in GUCY2D segregating with disease was observed. In the third pedigree compound heterozygous mutations, p.G104V and p.R124* in the gene RPE65 were observed to segregate with retinal degeneration. Analysis of exome sequence variants observed in the proband of the fourth pedigree detected one novel homozygous missense, potentially pathogenic change p.D2102E in the ABCA4 gene. Analysis of 100 ethnicity-matched controls did not detect the presence of the novel ABCA4 change indicating that this is a rare variant in Mexican population. Conclusions: Analysis of 15 patients from Mexico for known mutations identified previously reported mutations in the USH2A, GUCY2D and RPE65 genes. Exome sequencing of the proband of one pedigree reveled a novel homozygous mutation in the ABCA4 gene. In summary, screening patients with a diagnosis of LCA, RP, Usher syndrome, cone/rod dystrophy from Mexico identified four previously described mutations and one novel mutation in genes implicated in causing recessive retinal dystrophies. Commercial Relationships: Adda L. Villanueva, None; Mathieu Langlois, None; Ian Mongrain, None; Sylvie Provost, None; Géraldine Asselin, None; Marie-Pierre Dubé, None; Keerti Tadimeti, None; John Suk, None; Pooja Biswas, None; Radha Ayyagari, None

©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected].

ARVO 2015 Annual Meeting Abstracts Program Number: 2867 Poster Board Number: C0123 Presentation Time: 8:30 AM–10:15 AM Molecular investigation of a large UK cohort of early onset retinal dystrophy Sarah Hull1, Robert Henderson2, 1, Phillip Moradi1, Graham E. Holder1, Michel Michaelides1, Andrew Webster1, Donna Mackay3, 1, Gavin Arno1, Anthony T. Moore1, 2, Arun Dev Borman1. 1Moorfields Eye Hospital/UCL Institute of Ophthalmology, London, United Kingdom; 2Great Ormond Street Hospital, London, United Kingdom; 3 Department of Ophthalmology and Visual Sciences, Washington University in St Louis, St Louis, MO. Purpose: To molecularly investigate a consecutive series of 458 patients (382 famlies) with early-onset severe retinal dystrophy (EOSRD), including Leber Congenital Amaurosis, and to perform in-depth phenotyping to determine key clinical and molecular characteristics and relationships. Methods: Consecutive patients from two tertiary referral centres with a clinical diagnosis of EOSRD (onset less than 5 years of age) were recruited. Molecular investigations included candidate gene Sanger sequencing, arrayed primer extension (APEX), next generation sequencing (NGS) with a 105 retinal gene panel, and whole exomeanalysis. Clinical investigations included retinal imaging and electrophysiology. Results: Analysis has so far identified likely disease-causing variants in 197 families. Mutations in 38 genes have been idenitifed with the 5 most frequent being CRB1 (18%, n=35), RDH12 (14%, n=28), RPE65 (12%, n=24) CEP290 (10%, n=20) and GUCY2D (5%, n=9). Considering the 185 unsolved families, no molecular diagnosis was apparent despite whole exome sequencing in 16, NGS retinal panel screening in 2 and APEX in the remaining 167. These 185 families are the subject of further investigation using whole exome and whole genome sequencing. Phenotype-genotype correlations that were identified included nummular retinal pigmentation with macular thickening and loss of lamination in CRB1 related dystrophy; photoattraction, peripheral fine white retinal dots, and absent/markedly reduced autofluorescence in RPE65 disease; and relatively normal fundus appearances in both CEP290 and GUCY2D associated disease. Non syndromic manifestations of genes previously reported to have systemic manifestations were also observed. This included IFT140 related retinal dystrophy in a family with no skeletal or renal manifestations of IFT140 related Mainzer-Saldino syndrome and COL18A1 related retinal dystrophy in a patient with normal neuroradiological imaging, unexpected for Knobloch syndrome. Conclusions: The 5 most frequent molecular causes of EORD in this large cohort account for 59% of the genetically characterised families. Recognising key clinical features can help target molecular investigation but in atypical phenotypic presentations molecular diagnosis may only be achieved by NGS or whole-exome analysis. Early molecular diagnosis facilitates genetic counselling,including prognosis and is an essential prerequisite for gene-specific interventions. Commercial Relationships: Sarah Hull, None; Robert Henderson, None; Phillip Moradi, None; Graham E. Holder, None; Michel Michaelides, None; Andrew Webster, None; Donna Mackay, None; Gavin Arno, None; Anthony T. Moore, None; Arun Dev Borman, None Support: The National Institute for Health Research (UK) and Biomedical Research Centre at Moorfields Eye Hospital and the UCL Institute of Ophthalmology, The Foundation Fighting Blindness (USA), Fight For Sight, Moorfields Eye Hospital Special Trustees, Rosetrees Trust

Program Number: 2868 Poster Board Number: C0124 Presentation Time: 8:30 AM–10:15 AM A Novel CRX Mutation in a Family with Findings Suggestive of Benign Concentric Annular Macular Dystrophy Joseph F. Griffith, Meghan J. Marino, Elias I. Traboulsi. Ophthalmology, Cole Eye Institute, Shaker Heights, OH. Purpose: To present the phenotype of a mother and son initially given a diagnosis of benign concentric annular macular dystrophy (BCAMD) and later found to have a novel nonsense mutation in the cone-rod homeobox (CRX) gene (19q13.3). Methods: Patients underwent complete ophthalmic examinations. The son underwent sequencing of 131 retinal dystrophy genes. His mother was tested for the presence of the discovered sequence variations in two potentially causative genes CRX and BBS12 (4q27). Results: The son (Case I) presented at age 35 years for routine eye care. He had a family history of multiple males and females with early onset retinal disease. Over nine years of follow-up, his visual acuity (VA) remains at 20/20 - 20/25 in each eye with normal color vision. Ophthalmoscopy shows peripapillary atrophy and a bull’s eye macular lesion. His mother (Case II) presented at age 57 years with unilateral, progressive vision loss for nine months. She reported a prior VA of 20/30 in the right eye and on examination was 20/400 in that eye. Over five years of follow-up, VA fluctuated from 20/100 - 20/400 in the right eye and remains 20/25 in the left eye with decreased color vision (1/11, Ishihara Color Test). Ophthalmoscopy is similar to Case I except for a smaller area of preserved central retina. Fundus autofluorescence shows parafoveal hypofluorescence and an outer ring of hyperfluorescence. OCT demonstrates atrophy of the outer retina, subretinal excrescences, and segmental loss of the ellipsoid layer and the retinal pigment epithelium. BCAMD was the working diagnosis because of the characteristic retinal lesion, preserved VA, and presumed autosomal dominant inheritance. Genetic testing identified a novel nonsense mutation in CRX (p.Gln256stop:c.766C>T) in both patients. The son was also heterozygous for a missense mutation in BBS12 (p.Gln620Arg:c.1859A>G), which was not present in Case II. Conclusions: CRX mutations are associated with cone-rod dystrophy 2, Leber congenital amaurosis 7, retinitis pigmentosa, cone dystrophy, and a recently described adult-onset macular dystrophy. Genetic studies of the original BCAMD family demonstrated linkage to 6p12.3-q16 and no mutations in CRX. There is wide clinical heterogeneity resulting from CRX mutations, including phenotypes characterized by a bull’s eye retinal lesion and fairly well preserved visual acuity. Commercial Relationships: Joseph F. Griffith, None; Meghan J. Marino, None; Elias I. Traboulsi, Retrophin (C), Sonofi (C) Program Number: 2869 Poster Board Number: C0125 Presentation Time: 8:30 AM–10:15 AM Retinitis Pigmentosa or Sperm Dysfunction as the Only Signs of BBS2 Mutations Brian D. Perkins1, Meghan J. Marino1, Gayle J. Pauer1, John (pei wen) Chiang2, Glenn Lobo1, Joseph Fogerty1, Stephanie A. Hagstrom1, Elias I. Traboulsi1. 1Cole Eye Institute, Cleveland Clinic Foundation, Cleveland, OH; 2Molecular Diagnostics Laboratory, Casey Eye Institute, Portland, OR. Purpose: To describe a family with retinitis pigmentosa (RP) due to BBS2 mutations and to functionally characterize the mutant proteins. Methods: Fundus photographs, visual fields, and OCT images were obtained for the female proband and a male sibling. Molecular analysis via Next Generation Sequencing (NGS) found two BBS2 missense alleles, which were confirmed by Sanger Sequencing. In

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ARVO 2015 Annual Meeting Abstracts silico analysis of the mutant alleles was evaluated using Polyphen-2, pMut, and SIFT algorithms. Antisense morpholinos against zebrafish bbs2 were injected into 1-cell embryos and assessed for convergentextension defects. Embryos were also injected with in vitro synthesized mRNA for wild-type and mutant human BBS2 alleles to test allele function. Protein localization of Bbs2 proteins was examined by immunohistochemistry in transfected HEK293T cells. Results: A 41 year-old Caucasian female was diagnosed with RP and displayed significant visual field constriction, nyctalopia, and peripheral pigmentary changes, but no systemic problems. Her 39 year-old brother was asymptomatic and had been diagnosed with a low percentage of motile sperm and abnormal sperm morphology when he and his wife were evaluated for infertility. Additional examination showed mild peripheral bone spicule changes in his fundus. NGS analysis found 2 BBS2 variants, a p.R275X pathogenic allele and a p.P134R novel allele, which were determined to be inherited independently from each parent. Both mutant alleles failed to rescue a morpholino knockdown of the zebrafish bbs2 gene. Immunohistochemistry of V5-tagged Bbs2 proteins found that wildtype Bbs2 localized to the basal body, whereas both mutant proteins aggregated away from the cilium. Conclusions: This family highlights the clinical variability in patients with Bardet-Biedl Syndrome and describes a novel BBS2 mutation. Both mutant alleles are nonfunctional, yet only RP and sperm dysfunction were observed. The results confirm that mutations in BBS2 may cause nonsyndromic recessive RP, and that infertility may be the only problem in patients with mild asymptomatic retinopathy. Commercial Relationships: Brian D. Perkins, None; Meghan J. Marino, None; Gayle J. Pauer, None; John (pei wen) Chiang, None; Glenn Lobo, None; Joseph Fogerty, None; Stephanie A. Hagstrom, None; Elias I. Traboulsi, Retrophin (C), Sanofi (C) Support: NIH Grant EY017037; Foundation Fighting Blindness; Research to Prevent Blindness (unrestricted award); Research to Prevent Blindness Doris and Jules Stein Professorship Program Number: 2870 Poster Board Number: C0126 Presentation Time: 8:30 AM–10:15 AM Panel-based Next Generation Sequencing improves mutation detection in Cone Rod Dystrophy patients Zhongqi Ge1, 2, Eric Zaneveld1, 2, Vincent Sun3, Hui Wang1, 2, Richard G. Weleber4, Mark E. Pennesi4, Robert K. Koenekoop3, Ruifang Sui5, Rui Chen1, 2. 1Human Genome Sequencing Center, Baylor College of Medicine, Houston, TX; 2Molecular and Human Genetics, Baylor College of Medicine, Houston, TX; 3McGill Ocular Genetics Laboratory, McGill University Health Centre, Montreal, QC, Canada; 4 Ophthalmology, Casey Eye Institute, Portland, OR; 5Ophthalmology, Peking Union Med College, Peking, China. Purpose: Previous molecular diagnostic studies of Cone Rod Dystrophy had a rate of solution below 25%. In this study, we aim to carry out a comprehensive molecular diagnosis of a large CRD cohort by testing both known CRD genes and other retinal disease genes to increase our diagnostic accuracy and reveal new genotype-phenotype associations. Methods: 146 sporadic Cone Rod Dystrophy (CRD) and Cone Dystrophy (CD) patients were recruited from various ethnicities including Caucasian, Han Chinese, and African American. A custom retinal panel including all known CRD and CD genes was used for Next Generation Sequencing based genetic testing of these patients. Mutations identified were further Sanger validated and subjected to segregation tests when parent DNA was available. Results: Causative mutations in known CRD genes were found in 49 cases (33.6%), including 2 cases where mutations were found in syndromic CRD genes CNNM4 and ALMS1. Consistent with

previous studies, ABCA4 and RPGR were the most frequently mutated genes found in this cohort and accounted for 15 and 5 solved cases, respectively. Interestingly, we found genes that showed ethnic specificity. For example, mutations in PRPH2 and CNGA3 were only identified in Caucasians, while PROM1 mutations were exclusively found in Chinese patients. In addition, mutations in other nonsyndromic and syndromic retinal disease genes such as Retinitis Pigmentosa, Macular Dystrophy, and Bardet-Biedl syndrome were found in another 17 cases (11.6%). For example, CRB1 mutations, previously shown to cause Leber Congenital Amaurosis, were found in 4 patients. These findings may refine clinical diagnosis for some patients or implicate that new genes can cause CRD phenotype. Conclusions: This is the first comprehensive NGS based molecular diagnosis of CRD patients from multiple ethnicities involved. Our study demonstrated that panel-based NGS method is efficient, economic and sufficiently precise for molecular diagnosis CRD. Novel mutations identified will enrich our understanding of the genetic variations of CRD genes and lead to better molecular diagnosis of this disease. Acknowledgement: We would like to thank the patients the Research Group for their valuable contribution to this research. eyeGENE samples were obtained from the National Institutes of Health/ National Eye Institute - eyeGENE(r) - Protocol 06-EI-0236. Commercial Relationships: Zhongqi Ge, None; Eric Zaneveld, None; Vincent Sun, None; Hui Wang, None; Richard G. Weleber, None; Mark E. Pennesi, None; Robert K. Koenekoop, None; Ruifang Sui, None; Rui Chen, None Support: NIH 2T32EY007102-21A1 Program Number: 2871 Poster Board Number: C0127 Presentation Time: 8:30 AM–10:15 AM Leber’s Congenital Amaurosis and Retinitis Pigmentosa mutations in the domestic cat Leslie A. Lyons1, Christopher M. Reilly2, Ron Ofri4, David J. Maggs2, Barbara Gandolfi1, Hasan Alhaddad5, Robert A. Grahn3. 1Veterinary Medicine & Surgery, College of Veterinary Medicine, University of Missouri - Columbia, Columbia, MO; 2Surgical and Radiological Sciences, School of Veterinary Medicine, University of California - Davis, Davis, CA; 3Veterinary Genetics Laboratory, School of Veterinary Medicine, University of California - Davis, Davis, CA; 4Koret School of Veterinary Medicine, Hebrew University of Jerusalem, Jerusalem, Israel; 5College of Science, Kuwait University, Safat, Kuwait. Purpose: Two domestic cat breeds have been identified with autosomal recessive, early onset progressive retinal atrophies (PRAs). The Persian cat has clinical evidence of disease at 7 weeks of age with end-stage blindness by 16 weeks of age. Clinical onset in Bengal cats is at 12 weeks of age; but progression to end-stage blindness is slower and variable among individuals, with some cats still partially sighted at 2 years of age. Cross-breeding studies suggest a different locus for both forms of PRA. We hypothesized that genetic studies would identify DNA variants in genes causing PRA in these two cat breeds. Methods: Breeding colonies were established using donated cats handled according to institutional animal care and use protocols and ARVO guidelines. Vision status of all cats used in the genetic studies was established by board-certified veterinary ophthalmologists and pathologist. DNA was isolated using standard methods from whole blood collected from case and control cats, and was genotyped on the illumina Infinium 63K iSelect Cat DNA array. Association studies were performed using various methods. A trio of the Bengal and Persian cats each was whole-genome sequenced using an illumina

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ARVO 2015 Annual Meeting Abstracts HiSeq 100 bp paired end reads to approximately 30X genome coverage for each cat. Results: Association studies consistently localized the Persian and Bengal PRAs to cat chromosomes E1 (human chr. 17) and A3 (human chr. 20), respectively. Candidate genes for retinal degenerations within the regions were identified; however complete genetic sequences of the selected regions could not be obtained. Whole-genome sequencing of the Persian and Bengal trios identified candidate causal DNA variants. A polymorphism causing a premature stop codon was identified for the Persian PRA and a variant that alters a highly conserved amino acid was identified for the Bengal PRA, causing null and missense mutations, respectively. Genotyping of extended pedigrees of Persian, Bengal, and other feline populations supported these specific variants as causal for each PRA. Conclusions: Mutations causative for Persian and Bengal PRAs have been genetically identified. These diseases provide two new models for Leber’s Congenital Amaurosis and retinitis pigmentosa in a novel gene. Translational studies using these two feline models will support gene, stem cell and read-through based therapies. Commercial Relationships: Leslie A. Lyons, Veterinary Genetics Laboratory (C); Christopher M. Reilly, None; Ron Ofri, None; David J. Maggs, None; Barbara Gandolfi, None; Hasan Alhaddad, None; Robert A. Grahn, Veterinary Genetics Laboratory (E) Support: National Center for Research Resources R24 RR016094 and is currently supported by the Office of Research Infrastructure Programs OD R24OD010928, the Phyllis and George Miller Feline Health Fund, Center for Companion Animal Health, School of Veterinary Medicine, University of California – Davis (2007-38-FM, 2008-08-F); the Winn Feline Foundation (MT07-012, W12-022) (LAL) Program Number: 2872 Poster Board Number: C0128 Presentation Time: 8:30 AM–10:15 AM Concomitant presentation of novel mutations in NDP and LRP5 genes in a patient with suspected Norrie and FEVR diseases Mircea N. Coca1, Mohamed Soliman1, Emmanuel Chang2. 1 Ophthalmology and Visual Sciences, UTMB, Galveston, TX; 2Retina and Vitreous of Texas, Houston, TX. Purpose: To describe the clinical manifestation and genetic analysis of a patient with concomitant Norrie disease and Familial Exudative Vitreoretinopathy (FEVR) disease spectrum caused by two novel mutations in NDP and LRP5 genes, respectively. Methods: We report the case of a 6 weeks old full term Hispanic male who presented after his pediatrician noticed poor red reflexes bilaterally and a mildly enlarged left eye. A complete ophthalmologic examination was performed. The left eye exam was significant for proptosis, corectopia, elevated IOP, haziness and enlarged diameter of the left cornea. There were bilateral yellow, vascular membranes obscuring the view of the posterior segment. B scan ultrasound showed bilateral hyperechoic lesions connecting the posterior lens surface to the optic nerve head. Intraocular pressures measured 11 mmHg OD and 31 mmHg OS during an exam under anesthesia and handheld slit lamp examination showed shallow anterior chambers, prominent radial iris blood vessels, and posterior synechia bilaterally, with more advanced changes in the left eye. A Visual Evoked Potential test showed abnormal, extinguished responses to flash stimuli with cortical areas 17, 18, and 19 functional impairing. Hearing tests showed adequate hearing levels. Results: The chromosomal analysis established two novel gene mutations. The first is a hemizygous nonsense mutation c.211C>T (p.Q71*) in the exon 3 of X-linked NDP gene; other nonsense mutations have been reported on this exon in patients with Norrie disease.

The second is a homozygous unclassified mutation c.1130C>T (p.A377V) in the LRP5 gene. The alanine at amino acid position 377 of the LRP5 protein is highly conserved during evolution, so significance of this variant is currently unclear. Targeted parental sequence analyses indicates that the patient inherited c.1130C>T in the LRP5 gene from his asymptomatic father. The patient’s mother tested negative for any relevant mutations. Also a maternal uncle had a history of premature delivery, mental retardation, hearing loss and blindness requiring retina surgery, and glaucoma diagnosed just after birth. Conclusions: This is the first case reporting on two new concomitant mutations, one on X-linked NDP gene and the other LRP5 gene located on chromosome 11. LRP5 gene mutation is in a highly conserved region and its significance is currently unclear. Commercial Relationships: Mircea N. Coca, None; Mohamed Soliman, None; Emmanuel Chang, None Program Number: 2873 Poster Board Number: C0129 Presentation Time: 8:30 AM–10:15 AM SF3B2, a novel candidate gene for autosomal dominant retinitis pigmentosa, encodes a component of the U2 small nuclear ribonucleoprotein Caroline Van Cauwenbergh5, Kris Vleminckx4, Frauke Coppieters5, Marcus Karlstetter2, Thomas Langmann2, Gael Manes3, Christian P. Hamel3, Bart P. Leroy1, 5, Elfride De Baere5. 1Dept of Ophthalmology, Ghent University Hospital, Ghent, Belgium; 2Department of Ophthalmology, University of Cologne, Cologne, Germany; 3INM - Institut des Neurosciences de Montpellier Hôpital Saint-Eloi, INSERM U1051, Montpellier, France; 4Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium; 5Center for Medical Genetics, Ghent University, Ghent, Belgium. Purpose: Identification and functional characterisation of a novel candidate gene for autosomal dominant retinitis pigmentosa (adRP). Methods: Five affected and five unaffected individuals of a Belgian adRP family in which known adRP loci were excluded, were enrolled. They underwent genome-wide (GW) linkage analysis (BeadChip, Illumina). Whole exome sequencing was carried out in two affected individuals (HiSeq, Illumina; CLC bio). Segregation analysis of variants was done using Sanger sequencing and testing of 300 controls by HRM (LightScanner). Targeted resequencing of SF3B2 was performed (Miseq, Illumina) in 472 unrelated adRP patients. SF3B2 expression was tested using a commercial cDNA panel. Localization studies were carried out in 661W mouse cells using commercial anti-SF3B2 antibodies. Sf3b2 knockdown in Xenopus was done using targeted injection of a splicing blocking morpholino (MO) (GeneTools). Results: GW linkage analysis revealed two novel candidate loci with a maximum LOD score of 1.7. In the 11q13 region, a missense variant c.2417A>G p.(Tyr806Cys) was found in the SF3B2 gene encoding the splicing factor 3b, subunit 2. The Tyr residue is highly conserved, the Grantham distance between Tyr and Cys is 194, predictions suggest an effect on protein function. The change is predicted to disrupt a phosphorylation site. The variant co-segregates with adRP and is absent in 300 controls. No additional SF3B2 mutations were found in a large adRP cohort. Ubiquitous expression of SF3B2 was demonstrated in human tissues, including retina and RPE, and localization in perinuclear and nuclear areas was shown in 661W mouse cells. Targeted MO knockdown in Xenopus showed gross developmental anomalies affecting the retina. Rescue experiments are ongoing. Conclusions: SF3B2 was identified as a novel candidate gene for adRP. SF3B2 is required for binding of the U2 small nuclear ribonucleoprotein (snRNP) to the branchpoint and is involved in early

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ARVO 2015 Annual Meeting Abstracts spliceosome assembly. Interestingly, protein-protein interactions have been identified between SF3B2, SNRNP200 and PRPF8, the latter being two proteins implicated in adRP. So far, of the seven known adRP genes involved in splicing, six encode components of the U4/ U6-U5 triple small nuclear ribonucleoprotein (tri-snRNP) complex. Our study potentially involves other components of the spliceosome apart from the tri-snRNP complex in adRP. Commercial Relationships: Caroline Van Cauwenbergh, None; Kris Vleminckx, None; Frauke Coppieters, None; Marcus Karlstetter, None; Thomas Langmann, None; Gael Manes, None; Christian P. Hamel, None; Bart P. Leroy, None; Elfride De Baere, None Support: FRO to C.V.C. FWO 3G079711 to E.D.B., IAP project P7/43 (Belspo) to E.D.B. F.C. is a postdoctoral fellow, and B.P.L. and E.D.B. are senior clinical investigators of the FWO Program Number: 2874 Poster Board Number: C0130 Presentation Time: 8:30 AM–10:15 AM Presence of rd8 mutation does not effect the Lete-Onset Retinal Degeneration (L-ORD) phenotype in C57BL/6 mice with S163R Ctrp5/C1qtnf5 mutation Radha Ayyagari1, Bhubanananda Sahu1, Akhila Alapati1, John Suk1, Dirk-Uwe G. Bartsch1, Monica M. Jablonski3, Venkata R. Chavali2. 1 Ophthalmology, University of California San Diego, La Jolla, CA; 2 Ophthalmology, University of Pennsylvania, Philadelphia, PA; 3 Hamilton Eye Institute, University of Tennessee, Memphis, TN. Purpose: To study the influence of Crb1 c.3481delC (rd8) mutation on the retinal phentoype of the L-ORD mouse model with heterozygous S163R missense mutation in the C1Q-Tumor Necrosis Factor Related Protein-5 (C1QTNF5/CTRP5) gene. Methods: Mouse model for L-ORD was generated on C57BL/6J background and the presence of rd8 mutation was observed in this colony. To study the influence of the rd8 mutation on L-ORD phenotype, mouse lines carrying both the Ctrp5 S163R and the rd8 mutation (Ctrp5+/-;rd8/rd8), without the rd8 mutation (Ctrp5+/-;wt/wt); and wild type mice with and without the rd8 mutation (Wtrd8/rd8 and Wtwt/wt, respectively) were generated. Genotyping was carried out by allelic polymerase chain reaction (PCR) or sequencing. Retinal morphology was studied by fundus imaging, histology, light microscopy, electron microscopy and immunohistochemistry. Results: Genotype analysis of the mice in L-ORD mouse colony detected the rd8 mutation in both the homozygous or heterozygous states. Fundus imaging revealed an accelerated accumulation of AF spots with age in Ctrp5+/-;wt/wt. The number of AF spots was significantly increased with age in Ctrp5+/;wt/wt mice when compared to age matched controls. The presence of pseudorosette structures, the hallmark pathology in rd8 mice, was not observed in any genotypes studied. Further, the external limiting membrane was continuous in Ctrp5+/-;rd8/rd8 and Wtrd8/rd8 mice. The Ctrp5+/-;wt/wt mice developed characteristic L-ORD pathology including age-dependent accumulation of AF spots, development of prominent sub-RPE and basal laminar deposits and Bruch’s membrane abnormalities at an older age, while these changes were not observed in age-matched littermate Wtwt/wt mice. Conclusions: The Wtrd8/rd8 and Ctrp5+/-;rd8/rd8 mice raised on C57BL/6J did not develop early onset retinal changes that are characteristic of the rd8 phenotype supporting the hypothesis that manifestation of rd8-associated pathology is dependent on the genetic background. The lack of rd8- associated retinal pathology in the Ctrp5+/-;wt/ wt mouse model raised on the C57BL/6J background and the development of the L-ORD phenotype in these mice in the presence and absence of rd8 mutation suggests that the pathology observed in

Ctrp5+/-;wt/wt mice is primarily associated with the S163R mutation in the Ctrp5 gene. Commercial Relationships: Radha Ayyagari, None; Bhubanananda Sahu, None; Akhila Alapati, None; John Suk, None; Dirk-Uwe G. Bartsch, None; Monica M. Jablonski, None; Venkata R. Chavali, None Support: NIH-EY13198, NIH-EY21237, P30-EY22589, Foundation Fighting Blindness, Research to Prevent Blindness Program Number: 2875 Poster Board Number: C0131 Presentation Time: 8:30 AM–10:15 AM Disease-causing mutations in a cohort of autosomal dominant RP (adRP) families without detectable mutations in known adRP genes Lori S. Sullivan1, Sara J. Bowne1, Susan H. Blanton2, Daniel C. Koboldt3, Richard K. Wilson3, Rui Chen4, Feng Wang4, Dianna K. Wheaton5, David G. Birch5, Stephen P. Daiger1, 6. 1Human Genetics Center SPH, The Univ. of Texas HSC at Houston, Houston, TX; 2 Miami Institute for Human Genomics, Miller School of Medicine, Univ. of Miami, Miami, FL; 3The Genome Institute, Washington Univ. School of Medicine, St. Louis, FL; 4Human Genome Sequencing Center, Baylor College of Medicine, Houston, TX; 5 Retina Foundation of the Southwest, Dallas, TX; 6Ruiz Dept. of Ophthalmology and Visual Science, The Univ. of Texas HSC at Houston, Houston, TX. Purpose: To determine the cause of disease in a cohort of 64 families with a diagnosis of adRP but without disease-causing mutations detected using panel-based Sanger sequencing and retinal-capture next-generation sequencing (NGS). Methods: Unrelated probands in a cohort of 266 families with a diagnosis of adRP were tested by Sanger sequencing for mutations in known adRP genes and RPGR and RP2; probands without mutations were tested with retinal-capture NGS. Pathogenic mutations were identified in 76% of families. The remaining 64 families without detectable mutations were analyzed by a variety of methods including linkage mapping in suitably-large families, and whole-exome and whole-genome NGS. Potential disease-causing variants were tested in a panel of 200 adRP families not part of the original cohort. Results: Linkage mapping localized the disease gene in 6 families to one or a few chromosomal sites, largely non-overlapping sites across families. Known retinal-disease genes in or contiguous to each linkage region were excluded by high-resolution linkage testing and/ or sequencing. Whole-exome NGS was conducted in 10 or more affected members of the 6 families and in fewer members of 12 other families. Whole-genome NGS was conducted in 2 families. Rare variants tracking with disease were evaluated by bioinformatic methods and lists of potentially-pathogenic variants, and variants of unknown significance, were shared with other laboratories. A novel, dominant-acting mutation in RPE65 was identified in one family and a disease-causing mutation in a new adRP gene, HK1, was found in a second large family. From 10 to 100 rare variants were detected in the linkage regions of the remaining mapped families and numerous potentially-deleterious variants are tracking with disease in the smaller families. None of the rare variants detected are clearly pathogenic. Conclusions: Mutations in at least 21 genes are known to cause adRP; linkage mapping in families without mutations in known genes indicates that several adRP genes remain to be found. Wholeexome and whole-genome NGS are powerful tools for finding disease-causing mutations in retinal disease genes but establishing pathogenicity of rare variants in families without mutations in known genes is a difficult problem. Data sharing between laboratories with similar research interests is a helpful adjunct to genetics research.

©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected].

ARVO 2015 Annual Meeting Abstracts Commercial Relationships: Lori S. Sullivan, None; Sara J. Bowne, None; Susan H. Blanton, None; Daniel C. Koboldt, None; Richard K. Wilson, None; Rui Chen, None; Feng Wang, None; Dianna K. Wheaton, None; David G. Birch, None; Stephen P. Daiger, None Support: Hermann Eye Fund, The Foundation Fighting Blindness, Retinal Research Foundation, and NIH EY007142, EY022356, and EY018571 Program Number: 2876 Poster Board Number: C0132 Presentation Time: 8:30 AM–10:15 AM Recessive RHO mutation E150K and SAMD7 regulatory variants in a consanguineous family with retinitis pigmentosa Kristof Van Schil1, Marcus Karlstetter2, Alexander Aslanidis2, Bart P. Leroy1, 3, Frauke Coppieters1, Fanny Depasse4, Thomas Langmann2, Elfride De Baere1. 1Center for Medical Genetics, Ghent University and Ghent University Hospital, Ghent, Belgium; 2 Department of Ophthalmology, University of Cologne, Cologne, Germany; 3Department of Ophthalmology, Ghent University and Ghent University Hospital, Ghent, Belgium; 4Department of Ophthalmology, Queen Fabiola Children’s University Hospital, Brussels, Belgium. Purpose: To identify the genetic cause of a retinitis pigmentosa phenotype observed in a Turkish consanguineous family. Methods: The family consists of five sibs, two of which are affected. All family members underwent homozygosity mapping using the HumanCytoSNP-12 BeadChips (Illumina). Subsequent candidate gene screening was performed by Sanger sequencing. Functional analysis of non-coding SAMD7 variants was performed by luciferase assays in HEK293 cells and electroporation assays in mouse retinal explants with SAMD7 CBR-reported constructs (Hlawatsch et al. 2013). Results: Homozygosity mapping revealed several large homozygous regions, in which two interesting candidate genes were located, namely RHO and SAMD7. A homozygous RHO mutation (c.448G>A, p.E150K) was found in the two affected sibs, while all other sibs were heterozygous carriers. No coding SAMD7 mutations were found. Interestingly, sequencing of the SAMD7 promoter and an enhancer region in the two affected sibs revealed four homozygous variants located in the binding regions of the cone-rod homeobox (CRX) transcription factor. The variants are known SNPs, with a low minor allele frequency of 1,6 %. The first three SNPs are located in a CRX-binding region called CBR1, while the fourth SNP is located in CBR2. A potential regulatory effect of these SNPs on SAMD7 expression was assessed by in vitro luciferase assays in HEK293 cells and electroporation assays in mouse retinal explants, using constructs with either CBR1 or CBR2 apart, or in tandem. The combined CBR1/CBR2 mutated construct showed a significantly decreased SAMD7 reporter activity compared with the wild type (WT) CBR1/CBR2 construct, both in the cellular luciferase and mouse electroporation assays respectively. Conclusions: A rare recessive RHO mutation (E150K) was found in RP patients from consanguineous origin, consistent with previous reports (Kumaramanickavel et al., 1994; Azam et al., 2009 and Zhang et al., 2013). Moreover, functional analysis of four variants located in non-coding, CRX-binding regions of SAMD7, suggested a regulatory and synergistic effect of these upstream SNPs on SAMD7 expression. As Samd7 has recently been identified as a novel Crxregulated transcriptional repressor in retina (Hlawatsch et al., 2013), we hypothesize that these SAMD7 variants might have a modifying effect on the retinal phenotype observed in this family. Commercial Relationships: Kristof Van Schil, None; Marcus Karlstetter, None; Alexander Aslanidis, None; Bart P. Leroy,

None; Frauke Coppieters, None; Fanny Depasse, None; Thomas Langmann, None; Elfride De Baere, None Support: IWT doctoral grant to K.V.S. FWO11/KAN/013-31524611; FWO 3G079711, to E.D.B. FWO/KAN/1520913N to F.C. IAP project P7/43 (Belspo) to E.D.B. FRO to K.V.S. and E.D.B. FWO postdoctoral grant to F.C. FWO FKM to E.D.B. Program Number: 2877 Poster Board Number: C0133 Presentation Time: 8:30 AM–10:15 AM Mutations in MFSD8 can indeed cause a nonsyndromic recessive macular dystrophy. Mohammed E. Elasrag1, 2, Martin McKibbin3, James Poulter1, Chris Inglehearn1, Carmel Toomes1, Manir Ali1. 1Section of Ophthalmology and Neuroscience, Leeds Institute of Biomedical and Clinical Sciences, University of Leeds, Leeds, United Kingdom; 2Department of Zoology, Faculty of Science, Benha University, Benha, Egypt; 3 The Eye Department, St. James’s University Hospital, Leeds, United Kingdom. Purpose: To identify the genetic basis of macular dystrophy in three affected siblings of a non-consanguineous Caucasian family living in the UK. Methods: Whole-exome sequencing (WES) was performed using SureSelectXT Human V4 target enrichment reagent followed by paired-end sequencing on a HiSeq2500.The data files were processed on the Galaxy platform, aligned to the human reference genome (hg19/GRCh37) and the reported variants annotated using Annovar. Variants were excluded if they were outside the exon and flanking splice recognition sites, were synonymous or had a minor allele frequency >1% in the exome variant server database. Variants were confirmed by PCR and Sanger sequencing. Results: Genomic DNA from an affected family member was analysed by WES and the list filtered for prioritization of variants that are in the known RetNet genes. We identified 5 heterozygous and 1 homozygous variant though none segregated with the disease phenotype in the family. WES analysis of a second affected siblingand comparison of the full lists for common variantsidentified 15 homozygous and 4 genes with compound heterozygous variants. It was noted that mutations in one of these candidates MFSD8 (major facilitator superfamily domain-containing protein 8) had recently been reported in 2 families to cause nonsyndromic recessive macular dystrophy (Roosing et al., Ophthalmology, 2014). Sanger sequencing of the MFSD8 variants, c.1006G>C, p.E336Q and c.1394G>A, p.R465Q, in the UK family confirmed that all the affected members were indeed compound heterozygous for the mutations. These results are surprising since all publications to date on mutations in this gene except the one above showed that recessive MFSD8 mutations cause a severe multisystem lysosomal storage disorder, neuronal ceroidlipofuscinosis. Conclusions: This is the second study and only the third family describing biallelic MFSD8 mutations causing a nonsyndromic eye phenotype, and independently confirms the findings of the recent report.It is worth highlighting that one of these mutations, p.E336Q, exists in a heterozygous state in all three families and may act as a modifier of disease symptoms resulting in the less severe phenotype. Commercial Relationships: Mohammed E. Elasrag, None; Martin McKibbin, None; James Poulter, None; Chris Inglehearn, None; Carmel Toomes, None; Manir Ali, None Support: This research was supported by Egyptian government scholarship for Mr Mohammed Elasrag

©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected].

ARVO 2015 Annual Meeting Abstracts Program Number: 2878 Poster Board Number: C0134 Presentation Time: 8:30 AM–10:15 AM An augmented ABCA4 screen targeting non-coding regions reveals a deep intronic founder causal variant in Belgian Stargardt patients Miriam Bauwens1, Julie De Zaeytijd2, Nicole Weisschuh3, Susanne Kohl3, Françoise Meire4, Karin Dahan5, Fanny Depasse4, Frauke Coppieters1, Bart P. Leroy1, 2, Elfride De Baere1. 1Center for Medical Genetics Ghent, Ghent University, Ghent, Belgium; 2Ghent University Hospital, Department of Ophthalmology, Ghent, Belgium; 3 Molecular Genetics Laboratory, Institute for Ophthalmic Research, University of Tuebingen, Tuebingen, Germany; 4Department of Ophthalmology, Queen Fabiola Children’s University Hospital, Brussels, Belgium; 5Institut de Pathologie et de Génétique, Centre de génétique humaine, Gosselies, Belgium. Purpose: Autosomal recessive Stargardt disease (STGD1) is hallmarked by a large proportion of patients with a single heterozygous causative variant in the disease gene ABCA4. Braun et al. (2013) reported deep intronic variants of ABCA4, prompting us to perform an augmented screen in 131 Belgian STGD1 patients with one or no ABCA4 variant to uncover deep intronic causal ABCA4 variants. Methods: All 131 prescreened patients underwent targeted resequencing of four deep intronic ABCA4 regions using nextgeneration sequencing (NGS) (Miseq, Illumina). Haplotype analysis was performed on patients with variant c.4539+2001G>A (known as V4) and family members. Analysis and comparison of clinical and molecular data allowed us to establish phenotype-genotype correlations of V4. Data analysis was performed with CLC Bio. Filtering of non-coding variants was based on criteria such as minor allele frequency, conservation score, splicing predictions and regulatory potential.The cohort without an identified second mutation currently undergoes targeted NGS with a customized Haloplex panel that includes the entire ABCA4 gene and its regulatory domain, and the entire genomic regions of BEST1, RDH12 and PRPH2. Results: Our augmented screen revealed a second variant in 28.6% of cases. Twenty-six percent of these carry the same causal variant V4. Haplotyping in V4 carriers showed a common region of 63 kb, suggestive of a founder mutation. Genotype-phenotype correlations indicate a moderate-to-severe impact of V4 on the STGD1 phenotype. The remaining patients in whom no second ABCA4 mutation was identified undergo NGS of the entire genomic regions of ABCA4, BEST1, RDH12 and PRPH2, some of which are regulatory regions (~ 500 kb in total). Preliminary sequence data of five patients show that the standard filtering yields approximately 130 variants per patient that require further investigation. Conclusions: Causal variant V4 occurs in a high fraction of Belgian STGD1 patients and represents the first deep intronic founder mutation in ABCA4. This emphasizes the importance of augmented molecular genetic testing of ABCA4 in Belgian STGD1 patients. Finally, more extensive resequencing in the remainder of the STGD1 patients without an identified second ABCA4 mutation, will allow us to identify novel deep intronic and non-coding mutations and to establish a more definite molecular diagnosis. Commercial Relationships: Miriam Bauwens, None; Julie De Zaeytijd, None; Nicole Weisschuh, None; Susanne Kohl, None; Françoise Meire, None; Karin Dahan, None; Fanny Depasse, None; Frauke Coppieters, None; Bart P. Leroy, None; Elfride De Baere, None Support: FWO grant 1T9214N to M.B., FWO 3G079711 to E.D.B., FWO/KAN/1520913N to F.C

Program Number: 2879 Poster Board Number: C0135 Presentation Time: 8:30 AM–10:15 AM Undiagnosed retinal disease cases caused by mutations in noncanonical retinal disease genes Xia Wang, Yanming A. Feng, Richard A. Lewis, Victor Zhang, Jing Wang, Lee-Jun Wong. Molecular and Human Genetics, Baylor College of Medicine, Houston, TX. Purpose: Inherited retinal diseases are phenotypically and genetically heterogeneous. Patients with one clinical diagnosis may carry pathogenic mutations in disease genes that are not typically associated with that clinical diagnosis. An accurate molecular diagnosis can help to refine and improve the clinical diagnosis. Methods: We applied target capture next generation sequencing in a clinical diagnostic setting for the molecular diagnosis of 112 retinal disease patients, including 90 with retinitis pigmentosa (RP), 12 with Leber congenital amaurosis (LCA), and 10 with exudative vitreoretinopathy (FEVR) cases. For each case the coding exons and their 20 bp flanking regions of 207 eye disease genes were captured and sequenced. We first analyzed variants in canonical retinal disease for the corresponding patients, and then analyzed variants in the remaining eye disease related genes for the unsolved cases. Results: Pathogenic variants in canonical retinal disease genes were identified in 77% (69/90), 58% (7/12), and 30% (3/10) of RP, LCA, and FEVR cases respectively, suggesting high diagnostic yields. Among the remaining cases, pathogenic variants in noncanonical retinal disease genes were identified in 5 RP, 2 LCA, and 2 FEVR cases. For examples, one patient was suspected to have LCA but was found to carry compound heterozygous novel nonsense mutations in ALMS1. We later confirmed that this patient had hearing loss and mental retardation in addition to the blindness, which together resemble Alstrom syndrome. One patient tested negative for mutations in nonsyndromic RP genes was found to harbor a heterozygous reported mutation in GUCA1A, which has been associated with autosomal dominant cone dystrophy. Since this patient was 33 years old at the time of diagnosis, the disease may be at a late stage for clear distinction of the clinical differences. Another patient tested negative for mutations in FEVR genes was found to carry a hemizygous well known RS1 mutation. Since this patient had retinal detachment, his phenotype may fit the clinical diagnosis of retinoschisis. Conclusions: Our results highlighted the clinical and genetic heterogeneity of retinal diseases. To obtain a more accurate molecular diagnosis and increase the diagnostic yield, we need to sequence and analyze a larger set of related retinal disease genes. Commercial Relationships: Xia Wang, None; Yanming A. Feng, None; Richard A. Lewis, None; Victor Zhang, None; Jing Wang, None; Lee-Jun Wong, None Program Number: 2880 Poster Board Number: C0136 Presentation Time: 8:30 AM–10:15 AM Genotype-phenotype correlation in retinopathy associated with mitochondrial trifunctional protein disorders: Long-term followup of 21 cases Erin A. Boese, Nieraj Jain, Yali Jia, Catie Schlechter, Cary Harding, David Huang, Richard G. Weleber, Melanie Gillingham, Mark E. Pennesi. Oregon Health and Science University, Portland, OR. Purpose: We assess long-term effects of genotype and metabolic control on retinopathy severity in subjects with mitochondrial trifunctional protein disorders. Methods: This is a retrospective review of all patients evaluated at our institution from 1994 to 2014 with long-chain 3-hydroxyacylCoA dehydrogenase deficiency (LCHADD) or mitochondrial trifunctional protein deficiency (MTPD). All subjects underwent

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ARVO 2015 Annual Meeting Abstracts psychophysical testing and electroretinography, and select subjects also underwent multimodal imaging including optical coherence tomography (OCT) angiography. Subjects also participated in a diet optimization program with analysis of serum hydroxyacyl carnitines. Results: Twenty-one subjects were followed for a median of 5.5 years (range 0.0-20.2). Median age at the initial and final visit were 5.0 (range 0.4-17.7) and 10.5 (range 3.1-24.2) years, respectively. Three subjects (14.3%) had MTPD and 18 (85.7%) had LCHADD. All subjects with LCHADD carried at least one copy of the common mutation (1528G>C) in the HADHA gene, and 9 (50%) were homozygous for this mutation. Mean logarithm of the minimum angle of resolution (LogMAR) for LCHADD subjects was 0.20 ±0.25 at baseline and 0.42 ±0.40 at the final visit. Subjects with MTPD had excellent and stable BCVA, with mean final LogMAR 0.0 ±0.0. Despite extensive posterior pole disease, 88% of LCHADD patients had final LogMAR ≤0.30 in the better seeing eye. Subjects with LCHADD, but not MTPD, had a mean decrease in spherical equivalent of 0.40 ±0.37 diopters per year, demonstrating strong inverse correlation with age (ρ =-0.82 in homozygous subjects). OCT imaging demonstrated early outer retinal irregularity, with well demarcated regions of severe outer retinal and RPE atrophy at later stages with choriocapillaris loss by OCT angiography. Serial imaging demonstrated formation of outer retinal tubulations at transition zones, and these lesions were identified in all imaged LCHADD eyes. Conclusions: Long term follow up with multimodal imaging in this large cohort illustrates the worse ocular phenotype in LCHADD as compared to MTPD, with progressive chorioretinal atrophy, prominent outer retinal tubulations, and diffuse choriocapillaris loss. LCHADD subjects also had progressive myopia and declining visual function. Interestingly, most subjects retain reading vision in at least one eye. Commercial Relationships: Erin A. Boese, None; Nieraj Jain, None; Yali Jia, Optovue, Inc (F), Optovue, Inc (P); Catie Schlechter, None; Cary Harding, None; David Huang, Carl Zeiss Meditec, Inc (P), Optovue, Inc (F), Optovue, Inc (I), Optovue, Inc (P); Richard G. Weleber, None; Melanie Gillingham, None; Mark E. Pennesi, Sucampo (C) Support: Unrestricted grant from RPB, FFB Enhanced CDA (Pennesi), RPB CDA (Pennesi), 1K08 EY0231186-01 (Pennesi), R01 EY023285 (Jia, Huang), R01 EY024544 (Jia, Huang), DP3 DK104397 (Jia, Huang), RPB (Jia, Huang), CTSA grant (UL1TR000128) (Jia, Huang), FFB CDA (CF-CL-0614-0647OHSU) (Jain) Program Number: 2881 Poster Board Number: C0137 Presentation Time: 8:30 AM–10:15 AM A new mouse model of a late onset retinal degeneration (rd21) Bo Chang, Bernard FitzMaurice, Jieping Wang, Patsy M. Nishina. The Jackson Laboratory, Bar Harbor, ME. Purpose: Mouse models of inherited retinal degeneration are powerful tools to investigate the etiology of similar ocular diseases in humans. We have identified a new retinal degeneration model by routine screening of mouse strains through the Eye Mutant Resource program at The Jackson Laboratory. The aim of this study is to characterize the genetics and disease phenotype of this new model, retinal degeneration 21 (rd21) with a late, but rapidly progressive photoreceptor degeneration that is associated with retinal detachment. Methods: We characterized the clinical effects of this mutation using the Micron III in vivo bright field retinal imaging microscope with image-guided Optical Coherence Tomography (OCT), serial electroretinography (ERG) and histology, and performed genetic

linkage analysis to identify the chromosomal location of the mutation. Results: Mice homozygous for rd21 exhibit attenuated retinal vessels at five month of age and areas of depigmentation at six months of age. Histology and OCT at 5 months of age showed ~20-30% photoreceptor loss, with rapid progressive loss thereafter. By 7 months of age, there are no photoreceptors left and retinal detachment is observed by OCT. Electroretinograms (ERG) of rd21/ rd21 mice show near normal Rod ERG responses at 4 months of age, abnormal rod ERG responses (lower ERG a-wave, but near normal b-wave) at five month of age, with normal cone ERG responses within the same time frame. At 8 months of age, neither cone or rod ERGs waveforms are detectable. The inheritance pattern of the rd21 mutation is autosomal recessive. Linkage studies indicated that this new mutation maps to mouse Chromosome 19, with close linkage to a microsatellite marker, D19Mit61. Examination of candidates within the region, revealed an excellent candidate gene, rod outer segment membrane protein 1 (Rom1). Two other candidates mapping within the critical region are Best1 (bestrophin 1) and Stx3 (syntaxin 3). Conclusions: Late onset and rapidly progressive retinal function loss and degeneration combined with our genetic data suggest that this is a new spontaneous mutation not previously described in mouse. rd21 provides a novel mouse model for late onset photoreceptor degeneration associated with retinal detachment. Commercial Relationships: Bo Chang, None; Bernard FitzMaurice, None; Jieping Wang, None; Patsy M. Nishina, None Support: NIH Grant EY019943, EY011996 Program Number: 2882 Poster Board Number: C0138 Presentation Time: 8:30 AM–10:15 AM Leber congenital amaurosis with early-onset severe macular atrophy and optic atrophy is likely pathognomonic of NMNAT1 mutations Olivia Xerri1, Isabelle Perrault1, Sylvain Hanein1, Nathalie Delphin2, Lucas Fares-Taie1, Sylvie Gerber1, Olivier Roche3, Arnold Munnich1, Josseline Kaplan1, Jean-Michel Rozet1. 1Laboratory of Genetics in Ophthalmology (LGO), Institut Imagine, Paris, Paris, France; 2 Medical Genetics Department, Necker Enfants Malades Hospital, Paris, France; 3Ophtalmology department, Necker Enfants-Malades Hospital, Paris, France. Purpose: The nuclear nicotinamide mononucleotide adenyltransferase 1 (NMNAT1) encodes a homohexameric NADsynthesizing enzyme as well as a chaperone that protects against neuronal activity-induced degeneration. NMNAT1 mutations have been reported to cause a highly specific Leber congenital amaurosis (LCA) phenotype characterized by severe neonatal neurodegeneration of the central retina with early-onset optic atrophy. The purpose of the present study was to search for second NMNAT1 disease alleles in single heterozygote patients harboring the NMNAT1 phenotype. Methods: Sixteen sporadic cases and two sibs harboring single heterozygote NMNAT1 mutations were screened for copy number variations (CNV) using quantitative multiplex PCR of short fluorescent fragments (QMPSF) and qPCR digital droplet; and for mutations affecting regulatory elements or the splicing using Sanger sequencing of genomic DNA and/or lymphoblastoid cDNA. NMNAT1 haplotypes were constructed using SNP and microsatellite segregation analysis to search for shared ancestral alleles. Results: cDNA sequencing and/or CNV analysis allowed identifying a one-exon duplication in the two sibs, and 1 or 2-exon deletions in 4/17 unrelated patients. Sanger sequencing of non-coding NMNAT1 regions allowed identifying two rare variants possibly affecting a 5’ regulatory elements in 6/17 index cases. One out of the two variants was shared by 5 index cases originating from a French Indian

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ARVO 2015 Annual Meeting Abstracts Ocean Island (La Réunion). All five individuals harbored a common haplotype at the NMNAT1 locus supporting the transmission of an ancestral allele. Interestingly, none of the two variants could be detected at the cDNA level, suggesting down regulation or instability of the mRNAs harboring these changes. Two other 5’ changes were identified in 2/17 individuals which analysis is ongoing. Conclusions: Here, we report that at least 14/18 LCA individuals with single heterozygote NMNAT1 mutations carried a second disease allele undetected by Sanger sequencing of the NMNAT1 exome. This result suggests that severe neonatal neurodegeneration of the central retina with early-onset optic atrophy is pathognomonic of NMNAT1 mutations. In-depth molecular analysis of the gene and surrounding regulatory elements should be considered in all patients harboring this highly specific LCA phenotype Commercial Relationships: Olivia Xerri, None; Isabelle Perrault, None; Sylvain Hanein, None; Nathalie Delphin, None; Lucas Fares-Taie, None; Sylvie Gerber, None; Olivier Roche, None; Arnold Munnich, None; Josseline Kaplan, None; Jean-Michel Rozet, None Program Number: 2883 Poster Board Number: C0139 Presentation Time: 8:30 AM–10:15 AM Mutational analysis of TYR and OCA2 genes in four Chinese families with oculocutaneous albinism Yun Wang. Shenzhen Eye Hospital, Shenzhen, China. Purpose: Oculocutaneous albinism (OCA) is an autosomal recessive disorder. The most common type OCA1 and OCA2 are caused by homozygous or compound heterozygous mutations in the tyrosinase gene (TYR) and OCA2 gene, respectively.This study was to evaluate the molecular basis of oculocutaneous albinism in four Chinese families. Methods: We examined four non-consanguineous OCA families. The TYR and OCA2 genes of all individuals were amplified by polymerase chain reaction (PCR), sequenced and compared with a reference database. Results: Four patients, diagnosed as oculocutaneous albinism, presented with milky skin, white or light brown hair and nystagmus. Genetic analyses demonstrated that patient 1 was compound heterozygote for c.1037-7T.A, c.1037-10_11delTT and c.1114delG of TYR gene; patient 2 was heterozygote for c.593C>T and c.1426A>G of OCA2 gene, patient 3 and 4 was compound heterozygote of TYR gene (c.549_510delGT and c.896G>A, c.832C>T and c.985T>C). The heterozygous c.549_510delGT and c.1114delG alleles of TYR gene were two novel mutations. Interestingly, unaffected members in these pedigrees who carried c.1114delG of TYR gene or c.1426A>G of OCA2 gene presented with blond/brown hair and pale skin, but no ocular disorders when they were born, pigment was accumulated over time and with sun exposure. Conclusions: This study expands the mutation spectrum of oculocutaneous albinism. It is the first report that c.549_510delGT and c.1114delG of TYR gene were associated with OCA. The two mutations (c.1114delG of TYR gene and c.1426A>G of OCA2 gene) may responsible for part of clinical manifests of OCA. Commercial Relationships: Yun Wang, None Program Number: 2884 Poster Board Number: C0140 Presentation Time: 8:30 AM–10:15 AM Two Novel NYX Gene Mutations in The Chinese Families With X-Linked Congenital Stationary Night Blindness Ningdong Li. Tianjin Eye Institute, Tianjin, China. Purpose: To identify gene mutations in two Chinese families with X-linked congenital stationary night blindness (CSNB)

Methods: Two families with X-linked congenital stationary night blindness (CSNB) were recruited and patients underwent ophthalmological and electroretinography (ERG) examinations. Blood samples were collected and DNA was extracted. Two candidate genes of NYX and CACNA1F were directly sequenced and mutations analyzed. Two online programs of Polymorphism Phenotype (PolyPhen) and Sorting Intolerant From Tolerant (SIFT) were used to predict the potential impact of an amino acid substitution on the function of the protein. The molecular model were built using Swiss-model. Results: Most affected males showed variable degrees of myopia, a nearly normal fundus appearance, nystagmus and strabismus, as well as abnormal ERG with absence of rod b-wave and oscillatory potentials under the scotopic condition. Two NYX gene mutations were detected in the families, including a missense mutation of c.214A>C (p.N72H) and a small deletion of c.371_377del GCTACCT (p.Y125TfsX138), which were absent in 100 normal controls after sequencing of the NYX. Conclusions: These two novel mutations would expand the mutation spectrum of NYX gene and contribute to the study of the NYX gene’s molecular pathogenesis. Commercial Relationships: Ningdong Li, None Program Number: 2885 Poster Board Number: C0141 Presentation Time: 8:30 AM–10:15 AM Genetic influence of vascular patterns: the Minnesota Twins Reared Apart Study (MISTRA) Julian Tokarev1, Elena Bitrian2, Cheng Zhou1, Dara D. Koozekanani2, Erik J. Van Kuijk2, Nancy Segal4, Thomas Bouchard3. 1University of Minnesota Medical School, White Bear Lake, MN; 2Department of Ophthalmology, University of Minnesota, Minneapolis, MN; 3 Department of Psychology, University of Minnesota, Minneapolis, MN; 4Department of Psychology, California State University, Fullerton, CA. Purpose: To ascertain which retinal vascular features have strong genetic influences. The retinal vascular features examined herein are central retinal artery and vein equivalents (CRAE and CRVE) and arteriolar:venular ratio (AVR). Methods: Obtained from the Minnesota Study of Twins Reared Apart (MISTRA) registry, color fundus photographs of 66 pairs of monozygotic (MZA) and 40 pairs of dizygotic (DZA) twins reared apart were digitized from film slides at 600 DPI. The images were then graded using the software packages IVAN (Interactive Vessel Analyzer), v1.3 (courtesy of Dr. Nicola Ferrier of the Univ. of Wisconsin - Madison School of Engineering and the Dept. of Ophthalmology and Visual Sciences) and VAMPIRE (Vascular Assessment and Measurement Platform for Images of the Retina, available at vampire.computing.dundee.ac.uk). One grader performed all of the analysis with each software package. Results: Demographics: A total of 212 eyes from 53 pairs of twins (33 MZA pairs and 20 DZA pairs) were used. Average age (yrs) at imaging (mean ± stdandard deviation) for the included subjects was 38.0±13.8 MZA and 42.2±13.9 for DZA. 69.7% of the MZA and 65.0% of the DZA were female. Image Analysis: Of the 112 MZA images processed, 2 were ungradeable by IVAN. Of the 180 DZA images processed, 2 were ungradeable by IVAN and 1 by VAMPIRE. IVAN measurements showed no significant difference between MZA and DZA groups for mean CRAE, CRVE, or AVR. Comparison of average intertwin absolute differences in CRAE (MZA: 11.8±7.7, DZA: 20.3±13.5) showed significantly greater concordance between MZA pairs than DZA pairs (p=0.008). The same relationship was evident in a comparison of intertwin AVR differences (MZA: 0.0094±0.023,

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ARVO 2015 Annual Meeting Abstracts DZA: 0.052±0.047). The same comparison for CRVE (MZA: 10.8±9.5, DZA: 12.7±13.0) was nonsignificant between MZA and DZA pairs (p=0.29). Conclusions: In a unique cohort of twins reared apart, there is greater concordance between MZA twins than DZA twins for CRAE and AVR, but not CRVE. This suggests that CRAE and apparent AVR are more heritable than CRVE.

Figure 1. Automatic vascular segmentation performed with IVAN.

Figure. 2A Overview of fundus image with measurement zones in VAMPIRE. Figure. 2B Manual vascular measurement in VAMPIRE. Commercial Relationships: Julian Tokarev, None; Elena Bitrian, Vitreoretinal Research Foundation Grant (F); Cheng Zhou, None; Dara D. Koozekanani, None; Erik J. Van Kuijk, Research Preventing Blindness Grant (F), Vitreoretinal Research Foundation Grant (F); Nancy Segal, None; Thomas Bouchard, None Program Number: 2886 Poster Board Number: C0142 Presentation Time: 8:30 AM–10:15 AM OPN1LW/MW exon 3 haplotypes associated with Blue Cone Monochromacy disrupt exonic regulatory elements of splicing and can arise by gene conversion Elena Buena-Atienza1, Britta Baumann1, Nicole Weisschuh1, Klaus W. Ruether2, Susanne Kohl1, Bernd Wissinger1. 1Molecular Genetics Labor, Institute of Ophthalmic Research, Tuebingen, Germany; 2 Sankt Gertrauden-Krankenhaus, Berlin, Germany. Purpose: Cone Dysfunction syndromes such as Blue Cone Monochromacy (BCM) and Bornholm Eye Disease (BED) are caused by defects in the OPN1LW/MW (LW/MW) gene cluster. Subjects carrying a structurally intact opsin gene cluster and no ©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected].

ARVO 2015 Annual Meeting Abstracts known pathogenic point mutations were investigated for a novel third category of opsin mutations characterized by rare combinations of common single nucleotide polymorphisms (SNP) in exon 3 of LW/ MW. These rare haplotypes commonly termed by the amino acid combination they encode, such as LIAVA, may cause misplicing of the respective opsin gene. Methods: 16 affected subjects with a clinical diagnose of BCM or BCM-like were recruited and genotyped. Sequencing of LW/ MW exon 3 revealed novel haplotypes. The effect of various exon 3 haplotypes on transcript processing was tested applying minigene constructs heterologously expressed in HEK293 cells. Relative quantification of transcripts was performed by fragment analysis of FAM-labeled RT-PCR products. Genotyping of microsatellite markers and allelic cloning were performed to analyze the segregation and integrity of LW/MW in a family with evidence for gene conversion (GC). Results: In this cohort, 4 out of 13 different haplotypes analyzed in the minigene assay, LIAVA, LVAVA, MIAVA and a second MIAVA with one synonymous variant, resulted in a strong abundance of aberrantly spliced transcripts (90-100%). Further 4 haplotypes resulted in 40-80% of aberrantly spliced transcripts. Correlation of haplotype sequences with the minigene assay outcome suggests that the penultimate SNP in the haplotype, encoding for p.I178V is a strong exonic splicing silencer. In a case in which the phenotype switches from macular dystrophy with deuteranopia in the grandfather to BCM in the grandson, we found that intrachromosomal GC in the male germline introduced a deleterious LIAVA haplotype into LW. Conclusions: We found a variety of rare LW/MW exon 3 haplotypes associated with BCM or BCM-like phenotype and demonstrated fully or partially penetrant splicing defects caused by these variants. The 8 SNPs comprised in these haplotypes overlap with several exonic splicing silencer, enhancer and regulator signals involved in splicing regulation of LW/MW, and result in different outcomes when disrupted. Furthermore, we provide direct evidence that LIAVA haplotype in LW can be originated by intrachromosomal GC. Commercial Relationships: Elena Buena-Atienza, None; Britta Baumann, None; Nicole Weisschuh, None; Klaus W. Ruether, None; Susanne Kohl, None; Bernd Wissinger, None Support: FP7-PEOPLE-2012-ITN 317472 Program Number: 2887 Poster Board Number: C0143 Presentation Time: 8:30 AM–10:15 AM Identification of mutations in three retinal dystrophy genes within a single family: PRPF8, PRPH2 and USH2A Kaylie Webb-Jones1, Sara J. Bowne2, Lori S. Sullivan2, Stephen P. Daiger2, Feng Wang3, Rui Chen3, David G. Birch1, 4, Dianna K. Wheaton1, 4. 1Retina Foundation of the Southwest, Dallas, TX; 2 Human Genetics Center, Univ of Texas Health Science Ctr, Houston, TX; 3Molecular and Human Genetics, Baylor College of Medicine, Houston, TX; 4Ophthalmology, UTSW Medical Center, Dallas, TX. Purpose: To describe clinical and molecular characteristics of a family with seemingly incomplete penetrant, autosomal dominant retinitis pigmentosa (RP). Methods: Two female first cousins (20 & 22 yrs), previously diagnosed with RP with no reported hearing loss, and their respective mothers (48 & 53 yrs) underwent visual function exam and genotyping performed by capture next generation sequencing (NGS; 163 genes) and/or Sanger sequencing. Familial relationships were confirmed with short tandem repeat (STR) markers. Results: The proband (#8438) had VA=20/25 OU, Humphrey visual fields (HVF) were borderline reduced-to-mildly constricted, ffERG rod responses were reduced 88% in amplitude and delayed,

cone 30Hz flicker responses were reduced 76% and delayed, OCT imaging showed photoreceptor layer limited to the fovea and parafovea, no bone-spicule pigment was visible on fundus photography. Genetic analysis identified two known USH2A mutations in trans: p.Glu767Serfs*21 and p.Glu3448Lys. The proband’s mother (#9959) carries the USH2A p.Glu767Serfs*21 mutation and is asymptomatic with a normal appearing retina on OCT. The proband’s first cousin (#10228) had VA=20/32 OD, 20/40 OS, HVF constricted to A, p.Trp165* and c.131G>A, p.Ser44Asn. A gene-based, case-control association study using WES on a group of 18 probands with recessive macular dystrophy and 1,917 controls, highlighted DRAM2 as the most significantly enriched mutated gene; one subject was homozygous for c.362A>T, p.His121Leu and another was compound heterozygous for c.79T>C, p.Tyr27His and c.217_225del, p.73_75del. The clinical features and course of retinal degeneration were highly similar amongst affected cases from the four families. DRAM2 is a transmembrane protein that has previously been localized to lysosomes and has been implicated in inducing autophagy. Immunohistological analysis on retinal cross-sections showed DRAM2 localisation to photoreceptor outer segments where lysosomes are absent and suggests a novel role for DRAM2 in the retina. Conclusions: We have shown biallelic missense, nonsense and frameshift mutations in DRAM2 cause retinal dystrophy with early macular involvement. Our findings suggest that DRAM2 is essential for photoreceptor survival and further studies should provide insights into its role in photoreceptor outer segments. Commercial Relationships: Manir Ali, None; Mohammed E. Elasrag, None; Panagiotis Sergouniotis, None; Martin McKibbin, None; Vincent Plagnol, None; Zakia Abdelhamed, None; Declan McKeefry, None; Chris Inglehearn, None; Andrew Webster, None; Carmel Toomes, None Support: This work was supported by grants from NERC (Yorkshire branch), Macular Society UK (www.maculardisease.org), the UK National Institute for Health Research (Biomedical Research Centre, Moorfields Eye Hospital and Institute of Ophthalmology), RP Fighting Blindness and Fight For Sight (RP Genome Project GR586).

Program Number: 2889 Poster Board Number: C0145 Presentation Time: 8:30 AM–10:15 AM Recessive mutations in the polyglutamylase TTLL5, present in photoreceptor cells and spermatozoa, cause cone-rod degeneration and incompletely penetrant male infertility Nicola Bedoni1, Lonneke Haer-Wigman2, 3, Manir Ali4, Frans Cremers2, 3, Sten Andreasson5, Francis L. Munier6, Carlo Rivolta1. 1 Department of Medical Genetics, University of Lausanne, Lausanne, Switzerland; 2Department of Human Genetics, Radboud University Medical Center, Nijmegen, Netherlands; 3Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, Netherlands; 4Section of Ophthalmology & Neuroscience, Leeds Institute of Biomedical & Clinical Sciences, University of Leeds, Leeds, United Kingdom; 5Department of Ophthalmology, Lund University, Lund, Sweden; 6Jules Gonin Eye Hospital, Lausanne, Switzerland. Purpose: To identify the genetic causes of an atypical form of conerod dystrophy, sometimes associated with male infertility. Methods: We performed whole-genome-sequencing (WGS) in an affected male individual from a consanguineous family who was negative for mutations in genes known to be associated with retinal diseases. Further screening of TTLL5 exons and flanking intronic sequences was performed by direct Sanger sequencing. Immunofluorescence was used to detect TTLL5 within human control spermatozoa and murine retinal sections. Results: WGS of the index patient identified approximately 4 million DNA changes, which were reduced to 19 putative disease causing variants following the use of an internal in silico pipeline. Among these, we identified a homozygous frameshift mutation (c.1782del; p.Asp594Glufs*29) in the polyglutamylase gene TTLL5, confirming the recent published data that associate this gene to retinal dystrophy. Screening of TTLL5 exons and flanking intronic sequences in 49 patients with a diagnosis of cone or cone-rod dystrophy (33 from Sweden, 12 from Greece, as well as one English and three Dutch patients who displayed homozygosity for the chromosomal region containing TTLL5) identified four additional biallelic mutations in five individuals including another frameshift (c.2132_2133insGATA; p.Met712Ilefs*15), a nonsense (c.349C>T; p.Gln117*) and two missense (c.1627G>A; p.Glu543Lys and c.2266A>T; p.Ile756Phe) changes. Interestingly, two of the six patients with mutations in TTLL5 were infertile due to reduced sperm motility, a phenotype that is also displayed by Ttll5-/- male mice. Immunofluorescence with antiTTLL5 antibody in healthy human spermatozoa revealed that TTLL5 is localized at the base of the flagellum, near the centrioles, while in murine photoreceptors it is present at the basal body of the sensory cilium. Conclusions: Our observations suggest that TTLL5 is a component of both cilia and flagella and that its deficiency causes cone/cone-rod degeneration and incompletely penetrant male infertility. Commercial Relationships: Nicola Bedoni, None; Lonneke HaerWigman, None; Manir Ali, None; Frans Cremers, None; Sten Andreasson, None; Francis L. Munier, None; Carlo Rivolta, None Support: Swiss National Science Foundation Grant # 310030138346

©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected].

ARVO 2015 Annual Meeting Abstracts Program Number: 2890 Poster Board Number: C0146 Presentation Time: 8:30 AM–10:15 AM Severe Early-onset LCA-like Retinal Dystrophy due to Compound Heterozygous PRPH2 Mutations Dianna K. Wheaton1, 2, Kaylie Webb-Jones1, Sara J. Bowne3, Lori S. Sullivan3, Stephen P. Daiger3, Rui Chen4, Joost Felius1, 2, David G. Birch1, 2, Lori Dao5, Angela M. Scheuerle6. 1Retina Foundation of the Southwest, Dallas, TX; 2Ophthalmology, University of Texas Southwestern Medical Center, Dallas, TX; 3University Texas Health Science Center, Houston, TX; 4Baylor College of Medicine, Houston, TX; 5Pediatric Ophthalmology & Adult Strabismus, Dallas, TX; 6 Pediatrics, University of Texas Southwestern Medical Center, Dallas, TX. Purpose: To characterize the phenotype and genotype of an earlyonset, LCA-like dystrophy in an infant with congenital anomalies and developmental delay. Testing identified pathogenic mutations in three retinal dystrophy genes including the first observation of compound heterozygous PRPH2 mutations. Methods: At birth, the small for gestational age male exhibited micrognathia, a high palate, and obstructive apnea. Nystagmus, observed at age 6 months led to a provisional diagnosis of LCA. Dysphagia resulted in G-tube placement at 7 months. Exams at 9 months included a pediatric ophthalmology exam, a visual function assessment (Teller acuity, electroretinogram [ERG]), and a medical genetics consult to define the diagnosis. Retinal dystrophy genetic testing by next-generation sequencing was requested via research (163 genes) and diagnostic (53 genes) lab protocols. Results: The ophthalmology exam detailed a hypoplastic optic nerve (OD), trace retinal pallor, fine intermittent rotary nystagmus, and exotropia (OD). Teller acuity was abnormal for age at 20/145, rod ERG responses were normal whereas cone 30Hz flicker amplitudes were reduced 99% and delayed. The medical genetics exam detailed additional dysmorphic (microcephaly, prominent forehead, deep-set eyes, low set ears) and physical findings (hypotonia, Franceschetti oculo-digital sign, inability to sit/stand independently). Cardiovascular, pulmonary and abdominal exams were normal. Family history was unremarkable for anomalies or impairments of sight/hearing. Lab assays were normal for karyotype (46XY), SNP chromosome microarray, targeted sequencing of HADHA (LCHAD) and peroxisomal disorder analytes. Both retinal dystrophy genetic testing protocols identified two pathogenic PRPH2 mutations c.629C>G (p.Pro210Arg; dominant) and c.37C>T (p.Arg13Trp; digenic), and as well as a pathogenic ROM1 c.339dupG (p.Leu114fs; di-genic). A heterozygous, pathogenic USH2A mutation c.1606T>C was also identified. Parental testing confirmed the independent inheritance of the two PRPH2 mutations. Conclusions: PRPH2 mutations are typically associated with adultonset retinal dystrophy of variable phenotype ranging from pattern dystrophy to retinitis pigmentosa. The severe, early-onset phenotype observed in this PRPH2-induced retinal dystrophy is likely due to the PRPH2/ROM1 digenic mutations occurring concurrently with an additional dominant acting PRPH2 mutation. Commercial Relationships: Dianna K. Wheaton, None; Kaylie Webb-Jones, None; Sara J. Bowne, None; Lori S. Sullivan, None; Stephen P. Daiger, None; Rui Chen, None; Joost Felius, None; David G. Birch, None; Lori Dao, None; Angela M. Scheuerle, None Support: Foundation Fighting Blindness, NIH Grant EY007142

Program Number: 2891 Poster Board Number: C0147 Presentation Time: 8:30 AM–10:15 AM Absence of genotype-phenotype correlations in RPE65 gene mutations associated with autosomal recessive retinal dystrophy Daniel C. Chung1, Karmen Trzupek3, Jennifer Wellman2, Katherine High2. 1FM Kirby Ctr Molecular Ophth, Scheie Eye Institute, Philadelphia, PA; 2Spark Therapeutics, Inc, Philadelphia, PA; 3 InformedDNA, St. Petersburg, FL. Purpose: In many inherited disorders, genotype-phenotype correlations provide insight into onset of disease, rate of progression, and degree of severity. Both LCA2 and RP20 are caused by mutations in the RPE65 gene. We examined the various mutations in the RPE65 gene to determine whether genotype-phenotype correlations exist. Methods: Genotype data were obtained from individuals enrolled in the phase 1/2 and phase 3 gene therapy clinical trials at The Children’s Hospital of Philadelphia. These mutations were compared with mutations in the RPE65 gene from patients with autosomal recessive forms of inherited retinal dystrophy reported in the literature. Disease course and additional clinical data were evaluated. Results: Over 125 discrete mutations were identified in the RPE65 gene in individuals with inherited retinal disease. These include missense and nonsense point mutations, frameshift mutations resulting from small insertions and deletions, and splice site mutations. Patient diagnoses included Leber congenital amaurosis (LCA) type 2, early onset retinal dystrophy (EORD), and early onset retinitis pigmentosa (RP) type 20. Several recurrent mutations were noted across patients with LCA as well as later onset retinal dystrophies, including the missense mutation Tyr368His and the common splice site mutation in intron 1 (IVS1+5G>A). Nonsense and frameshift mutations were also described in patients across all diagnosis groups. Conclusions: In the cohort of subjects reviewed in this study, no clear genotype-phenotype correlations emerged. The distinction between RP20, LCA2, and other clinical diagnosis given to those with autosomal recessive RPE65 mutations appears to be based primarily on clinical symptoms and age of onset, and unrelated to underlying genotype. Possible factors linked to this lack of correlation include effects of modifier genes, wide differences among clinicians in assignment of clinical classification, or other causes. Commercial Relationships: Daniel C. Chung, None; Karmen Trzupek, Spark Therapeutics, Inc (C); Jennifer Wellman, Spark Therapeutics, Inc (E), Spark Therapeutics, Inc (I); Katherine High, Spark Therapeutics, Inc (E), Spark Therapeutics, Inc (I), Spark Therapeutics, Inc (S) Program Number: 2892 Poster Board Number: C0148 Presentation Time: 8:30 AM–10:15 AM Biallelic mutations in RBP3 encoding interphotoreceptor binding protein (IRBP) cause an early onset retinal dystrophy and high myopia Gavin Arno1, 2, Sarah Hull1, 2, Anthony G. Robson2, 3, Graham E. Holder2, 3, Andrew Webster1, 2, Vincent Plagnol4, Anthony T. Moore1, 2 1 . Inherited Eye Diseases, UCL Institute of Ophthalmology, London, United Kingdom; 2Moorfields Eye Hospital, London, United Kingdom; 3Visual Neuroscience, UCL Institute of Ophthalmology, London, United Kingdom; 4University College London Genetics Institute, London, United Kingdom. Purpose: To present a detailed clinical and molecular study of four patients from two consanguineous families with a similar childhoodonset retinal dystrophy resulting from novel biallelic nonsense mutations in RBP3. Methods: Four children with mutations in RBP3 encoding interphotoreceptor binding protein (IRBP) were ascertained by whole

©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected].

ARVO 2015 Annual Meeting Abstracts exome sequencing and subsequent direct Sanger sequencing. Detailed phenotyping was performed including full clinical evaluation, pattern and full-field electroretinography (PERG; ERG), fundus photography, fundus autofluorescence (FAF) imaging and spectral domain optical coherence tomography (OCT). Results: Two novel biallelic nonsense mutations (c.1530T>A ; p.Y510* and c.3454G>T ; p.E1152*) in RBP3 were identified in four patients from two families. All four patients had a similar, unusual retinal dystrophy characterised by childhood-onset high myopia and an unremarkable fundus appearance. The ERGs showed generalised rod and cone dysfunction in all cases, with greater rod involvement in three. PERG P50 ranged normal to undetectable, indicating variable macular involvement. FAF imaging showed multiple paracentral foci of low density in one patient and patchy increased FAF in the region of the vascular arcades in another. The OCT showed loss of outer retinal bands over eccentric macular areas in all 4 cases. Conclusions: This report is the first to describe the retinal dystrophy in children caused by biallelic RBP3 mutations. The retinal phenotype is characterised by early onset, normal fundi other than high myopia, and generalized retinal dysfunction with variable macular involvement. Mutations in RBP3 should be considered in children with high myopia and retinal dystrophy, particularly when there are minimal fundus changes. Commercial Relationships: Gavin Arno, None; Sarah Hull, None; Anthony G. Robson, None; Graham E. Holder, None; Andrew Webster, None; Vincent Plagnol, None; Anthony T. Moore, None Support: Fight for Sight, Rosetrees Trust Program Number: 2893 Poster Board Number: C0149 Presentation Time: 8:30 AM–10:15 AM TRNT1 MUTATIONS LINKED TO EARLY ONSET AUTOSOMAL RECESSIVE RETINITIS PIGMENTOSA Tasneem P. Sharma1, Robert F. Mullins1, Todd E. Scheetz1, Jessica Penticoff1, Malia Collins1, Trudi Westfall2, Jenna Barnes2, Diane C. Slusarski2, Budd Tucker1, Edwin M. Stone1. 1Ophthalmology & Visual Sciences, Stephen A. Wynn Institute for Vision Research, Carver College of Medicine, University of Iowa, Iowa City, IA; 2Biology, University of Iowa, Iowa City, IA. Purpose: Retinitis pigmentosa (RP), a heterogeneous group of monogenic disorders, is characterized by a variety of symptoms including night blindness and progressive death of photoreceptors. Through whole-exome sequencing, we recently identified novel mutations in the tRNA Nucleotidyl Transferase, CCA-Adding, 1 (TRNT1) gene in a proband displaying early-onset RP. Mutations in TRNT1 have been previously implicated in congenital sideroblastic anemia. The purpose of this study was 1) to demonstrate that TRNT1 mutations are causative of nonsyndromic autosomal recessive RP and 2) to elucidate the pathophysiologic mechanism of newly identified TRNT1 mutations in photoreceptor cell dysfunction and death. Methods: Next generation whole exome sequencing was used to identify potential disease-causing mutations in a proband with clinically diagnosed early onset autosomal recessive RP. Zebrafish, human induced pluripotent stem cells (iPSC), gene addition transfer, and CRISPR/Cas9-mediated genome editing technologies were used to confirm pathogenicity and develop potential treatment strategies. Results: Whole exome sequencing revealed two potential diseasecausing mutations in the gene TRNT1. Morpholino-mediated knockdown of TRNT1 in zebrafish caused a visual function defect that could in part be rescued by injection of wild-type RNA. Dermal fibroblasts from the proband were expanded and targeted for iPSC generation. IPSC pluripotency was confirmed using standard rt-PCR, immunoblotting, immunocytochemistry and teratoma formation. The genetic mutations identified via whole exome sequencing were

confirmed to alter retinal TRNT1 transcript and cause loss of fulllength TRNT1 protein in iPSC-derived retinal progenitor cells. Conclusions: Mutations in TRNT1 are capable of causing early onset, non-syndromic autosomal recessive RP. We have generated patientspecific iPSC-derived retinal cells to model disease in vitro. These cells will allow us to learn more about the pathophysiology and test multiple avenues of treatment for TRNT1-associated RP. Commercial Relationships: Tasneem P. Sharma, None; Robert F. Mullins, None; Todd E. Scheetz, None; Jessica Penticoff, None; Malia Collins, None; Trudi Westfall, None; Jenna Barnes, None; Diane C. Slusarski, None; Budd Tucker, None; Edwin M. Stone, None Support: Stephen A. Wynn Institute for Vision Research, NIH - Directors New Innovator Award DP2-OD007483-01, NEI RO1EY024605, NEI RO1-EY016822, Grousbeck Family Foundation, Foundation Fighting Blindness Center, Research to Prevent Blindness and Shramek Research Fund, The Elmer and Sylvia Sramek Charitable Foundation Program Number: 2894 Poster Board Number: C0150 Presentation Time: 8:30 AM–10:15 AM Mutation in EYS gene causes variable expressivity, ranging from Leber Congenital Amaurosis to adult onset Retinitis Pimentosa. Autofluorescence Study. Veronika Vaclavik1, 2, Francis L. Munier1, Daniel F. Schorderet1, Hoai V. Tran1. 1Hopital Ophtalmique Jules Gonin, Hosp Optalmique Jules Gonin Lausanne, Lausanne, Switzerland; 2service d’Ophtalmology, Hôpital Cantonal, Fribourg, Switzerland. Purpose: To determine genotype/phenotype correlation of EYS gene related autosomal recessive retinitis pigmentosa. The purpose of this study is to report a variable expressivity and an unusual appearance of fundus autofluorescence (AF). Methods: Six affected patients from 6 families underwent complete ophthalmological examination, inluding ISCEV standard full field ERGs, fundus photography, autofluorescence imaging (AF), optical coherence tomography (OCT) and kinetic visual field. Comprehensive gene screening was performed using a combined approach of different generation of DNA microarray analysis and direct sequencing. Results: Four novel mutations and two previously described mutations in EYS were identified in 6 families. The youngest affected patient was diagnosed with LCA during the first years of his life. He had nystagmus and VA was less than 3/60. Another young patient was diagnosed with early onset retinal dystrophy aged 4. Fundus examination revealed a range of retinal disorder from normal to optic nerve pallor, attenuated arterial caliber and bone spicule-like pigment deposits. In all patients, AF showed a double hyperfluorescent ring, in 4 patients a third ring was visible on wide field imaging. Conclusions: Mutations in Eys gene causes retinitis pigmentosa as well as LCA and Early Onset Retinal Dystrophy, previously unreported. A double and triple hyperfluorescent ring on AF is an uncommon observation and might be a specific clinical finding. Further analysis and a longitudinal study is required to determine the significance of those rings. Commercial Relationships: Veronika Vaclavik, None; Francis L. Munier, None; Daniel F. Schorderet, None; Hoai V. Tran, None

©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected].

ARVO 2015 Annual Meeting Abstracts Program Number: 2895 Poster Board Number: C0151 Presentation Time: 8:30 AM–10:15 AM Recurrence of identical combinations of mutations in MFSD8 or POC1B in persons with cone dysfunctions due to founder effects and putative functional constraints Riccardo Sangermano1, 2, Susanne Roosing5, L I. Van Den Born3, Carel C. Hoyng4, Frans Cremers1, 2, Lonneke Haer-Wigman1, 2. 1 Human Genetics, Radboud University Medical Center, Nijmegen, Netherlands; 2Radboud Institute for Molecular Life Sciences, Radboud university medical center, Nijmegen, Netherlands; 3The Rotterdam Eye Hospital, Rotterdam, Netherlands; 4Department of Ophthalmology, Radboud University Medical Center, Nijmegen, Netherlands; 5Department for Pediatric Brain Diseases, The Rockefeller University, New York, NY. Purpose: We previously identified MFSD8 or POC1B compound heterozygous variants in non-syndromic cone dystrophy (CD) cases. Both genes are implicated in syndromic phenotypes: MFSD8 in Batten disease and the POC1B ortholog poc1b in a syndromic zebrafish ciliopathy. This suggest that non-syndromic CD patients carrying MSFD8 or POC1B variants have residual activity of the respective proteins, due to the hypomorphic character of the identified variants. In order to establish genotype-phenotype correlation models we searched for additional non-syndromic CD cases harboring mutations in MFSD8 or POC1B. Methods: Whole exome sequencing (WES) was performed in 30 Dutch CD probands and Sanger sequencing was used for segregation analysis. An extensive clinical examination was conducted for persons harboring MFSD8 or POC1B variants. Haplotype analyses were performed using single nucleotide polymorphisms (SNPs) identified in WES data and selective SNP genotyping. Sequencing of MFSD8 exons 11 and 12 was performed in 235 retinal dystrophy probands and 300 controls. Results: For MFSD8, WES identified a Dutch CD case carrying compound heterozygous variants (p.K333Kfs*3; p.E336Q) described in another CD case (Roosing et al., Ophthalmology, 2014). Similarly, POC1B compound heterozygous variants (c.810+1G>T; p.Q67del) were identified in two unrelated CD cases (Roosing et al., Am J Hum Genet., 2014). For both genes, the affected cases carried identical haplotypes encompassing a region of ~ 2.8 or 1.5 Mb respectively. Besides, the MFSD8 p.E336Q variant was found in combination with p.E381* in five siblings with CD and in a heterozygous state in 1/300 Dutch controls and in 5/235 CD cases. Conclusions: We determined shared haplotypes in two Dutch cases with the same MFSD8 compound variants and in two cases with the same POC1B compound heterozygous variants, suggesting the presence of founder alleles for these genes. The MFSD8 p.E336Q mutation was found in all three CD families but not in 35 Batten disease probands reported earlier, indicating that it is a hypomorphic variant. Interestingly, all MFSD8 variants found in non-syndromic CD cases are located in the last three exons, which are jointly present or absent in MFSD8 isoforms. These findings suggest that both functional constraints and founder effects of mutations led to the recurrence of these genetic variants. Commercial Relationships: Riccardo Sangermano, None; Susanne Roosing, None; L I. Van Den Born, None; Carel C. Hoyng, None; Frans Cremers, None; Lonneke Haer-Wigman, None Support: EyeTN Grant agreement no.: 317472

Program Number: 2896 Poster Board Number: C0152 Presentation Time: 8:30 AM–10:15 AM The distinct clinical features of adult-onset retinal dystrophy associated with carboxyl mutations in the RPGR-ORF15 gene Rola Ba-Abbad1, 2, Panagiotis Sergouniotis1, 3, Anthony T. Moore1, 2 , Michel Michaelides1, 2, Graeme C. Black4, Simon C. Ramsden4, Anthony G. Robson1, 2, Graham E. Holder1, 2, Vincent Plagnol5, Andrew Webster1, 2. 1Institute of Ophthalmology, University College London, London, United Kingdom; 2Moorfields Eye Hospital, London, United Kingdom; 3University of Manchester, Manchester, United Kingdom; 4Manchester Centre for Genomic Medicine, Institute of Human Development, Manchester, United Kingdom; 5 UCL Genetics Institute, London, United Kingdom. Purpose: Mutations in the carboxyl end of the retinitis pigmentosa GTPase regulator (RPGR) exon 15 (ORF15) gene are usually associated with severe rod-cone dystrophy. This observational case series describes the distinct cone and cone-rod dystrophy phenotype associated with mutations in the carboxyl end of RPGR-ORF15. Methods: Probands who presented with signs and symptoms in keeping with macular or cone/cone-rod dystrophy, and a recessive inheritance pattern were included. Clinical assessment included fundus autofluorescence (AF); optical coherence tomography (OCT); and pattern (PERG) and full-field electroretinography (ERG). Leukocyte DNA from 2 simplex males was analyzed using highthroughput sequencing (exon capture by SureSelectXT Human All Exon V5, Agilent; sequencing by HiSeq2000, Illumina). Six further males with similar clinical features and 2 female carriers were analysed following PCR of the 3’ end of ORF15 using dideoxynucleotide Sanger sequencing. Results: Seven unrelated probands, and 1 related hemizygous male, were found to have 5 putative mutations in the carboxyl end of ORF15: (c.3092delA; c.3096_3097delGG; c.3178_3179delAG; c.3317dupA; c.3320_3323delCAGT). All were myopic and presented with adult-onset reduction of visual acuity (age 32-44, median 37 years; 20/30-20/600) and defective color vision. Fundus examination showed variable degrees of macular atrophy. There were bilateral paracentral rings of increased macular AF. The OCT showed loss of outer retinal bands within the AF rings. PERG and ERG showed macular and generalized cone dysfunction in all 8 patients, with additional rod dysfunction in 7 patients. Both carriers did not show significant fundus or ERG abnormalities. Conclusions: Male patients with retinal dystrophy associated with RPGR mutations usually manifest severe rod-cone dystrophy. However, those with mutations close to the 3’ end of ORF15 can have a less severe adult-onset cone or cone-rod dystrophy. This phenotype appears to occur with frameshifting mutations at or 3’ to c.3092 of the retinal transcript (NM_001034853), consistent with previous reports. This suggests that normal photoreceptor function, particularly in the cones, requires an intact sequence carboxyl to the polyglutamate repeat. Such cases are good candidates for gene-replacement therapy as early intervention may arrest progression before the onset of significant visual loss. Commercial Relationships: Rola Ba-Abbad, None; Panagiotis Sergouniotis, None; Anthony T. Moore, None; Michel Michaelides, None; Graeme C. Black, None; Simon C. Ramsden, None; Anthony G. Robson, None; Graham E. Holder, None; Vincent Plagnol, None; Andrew Webster, None

©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected].

ARVO 2015 Annual Meeting Abstracts Support: Supported by grants from the Macular Disease Society; the Foundation for Fighting Blindness; the National Institute for Health Research; Biomedical Research Centre at Moorfields Eye Hospital; National Health Service Foundation Trust and UCL Institute of Ophthalmology; Fight for Sight, RP Fighting Blindness, Moorfields Eye Hospital Special Trustees. RB is supported by an educational scholarship from King Saud University, the Ministry of Higher Education, Riyadh, Saudi Arabia. Program Number: 2897 Poster Board Number: C0153 Presentation Time: 8:30 AM–10:15 AM Phenotype and progression of retinal degeneration in patients nullizygous for ABCA4 Ana Fakin1, 2, Anthony G. Robson1, 2, Graham E. Holder1, 2, Kaoru Fujinami3, 6, Rando Allikmets4, 5, Jana Zernant4, Anthony T. Moore1, 2, Michel Michaelides1, 2, Andrew Webster1, 2. 1Institute of Ophthalmology, UCL, London, United Kingdom; 2Moorfields Eye Hospital, London, United Kingdom; 3Department of Ophthalmology, Keio University, School of Medicine, Tokyo, Japan; 4Department of Ophthalmology, Columbia University, New York, NY; 5Department of Pathology and Cell Biology, Columbia University, New York, NY; 6 National Institute of Sensory Organs, Tokyo Medical Center, Tokyo, Japan. Purpose: To describe the phenotype and progression of patients with severe ABCA4 retinopathy. Methods: A retrospective study was undertaken of 29 patients from 25 families harboring two likely-null ABCA4 mutations including stop mutations (25 alleles), frame-shifting indels (7 alleles) and splice-site mutations (18 alleles). Consecutive corrected visual acuity (VA) of the better eye, equal or above a threshold of 3/60 (1.3 LogMAR), were used for Kaplan-Meier survival analysis. Fundus autofluorescence imaging (FAF) was performed on the central 30 x 30 degrees (78 mm2) of the retina (Spectralis, Heidelberg, Germany). Regions of reduced fundus autofluorescence (FAF) were measured using RegionFinder tool included in the imaging software and plotted against age. Pattern and full-field electroretinography (PERG; ERG) were performed (N=19) and the results classified according to the published data1. Results: There were 20 male and 9 female patients with median age at onset at 8 years (range 4 – 29). Median age at their last visit was 21 years (range 6 - 75); median VA of the better eye was 3/60 (range 6/36 – light perception). VA correlated significantly with age (Spearman’s correlation, r = 0.61, p < 0.001); survival analysis showed that VA ≥ 3/60 in the better eye was retained in 50 % of patients until the age of 38 years. Areas of reduced FAF were observed in 96 % (22/23) patients. The median total area of reduced FAF at the last imaging was 9 mm2 (range 0 – 72 mm2). There was a strong correlation between size of reduced FAF and age (Pearson’s correlation, r = 0.89, p < 0.001). In 8 patients with repeated imaging at median follow up of 5 years the median rate of enlargement was 1.2 mm2 per year. The PERG and ERG data showed evidence of macular dysfunction with generalized cone and rod system involvement (group 3) in all. Conclusions: Nullizygosity for ABCA4 is associated with a relatively homogenous phenotype characterized by early onset of visual loss, rapid progression of central atrophy and concomitant peripheral retinal involvement. The description of progression rates in this disorder will inform the design and interpretation of novel treatment trials. Reference: 1. Lois N, et al. Phenotypic subtypes of Stargardt macular dystrophyfundus flavimaculatus. Arch Ophthalmol. 2001.

A. Visual acuity in correlation with age. B. Expansion of reduced FAF in 5 years in a patient homozygous for p.Y1027X mutation. Commercial Relationships: Ana Fakin, None; Anthony G. Robson, None; Graham E. Holder, None; Kaoru Fujinami, None; Rando Allikmets, None; Jana Zernant, None; Anthony T. Moore, None; Michel Michaelides, None; Andrew Webster, None Support: NIHR grant, NIH/NEI EY021163 and NIH/NEI EY019861 Program Number: 2898 Poster Board Number: C0154 Presentation Time: 8:30 AM–10:15 AM An intronic deletion in the PROM1 gene leads to autosomal recessive cone-rod dystrophy Tamar Ben-Yosef1, Osnat Eidinger1, Rina Leibu2, Hadas Newman3, Leah Rizel1, Ido Perlman4, 3. 1Genetics Dept - Faculty of Med, Technion, Haifa, Israel; 2Ophthalmology, Rambam Medical Center, Haifa, Israel; 3Ophthalmology, Tel-Aviv Medical Center, Tel Aviv, Israel; 4Physiology & Biophysics, Faculty of Medicine-Technion, Haifa, Israel. Purpose: To investigate the genetic basis for autosomal recessive cone-rod dystrophy (CRD) in a consanguineous Israeli Jewish family. Methods: Patients underwent a detailed ophthalmic examination, including funduscopy, electroretinography (ERG), visual field testing and optical coherence tomography (OCT). Genome-wide homozygosity mapping using a SNP array was performed to identify homozygous regions shared among two of the affected individuals. Mutation screening of the underlying gene was carried out by direct sequencing. In silico and in vitro analyses were used to predict the effect of the identified mutation on splicing. Results: The affected family members are three siblings suffering from progressive visual deterioration, glare, deficient color vision and night vision abnormalities. Visual field tests revealed central scotomas of different extension. Both cone and rod ERG responses were reduced, with cones being more severely affected. Homozygosity mapping revealed several homozygous intervals shared among two of the affected individuals. One of them included the PROM1 gene. Sequence analysis of the 26 coding exons of PROM1 in one affected individual revealed no mutations in the coding sequence or in intronic splice-sites. However, in intron 21, proximate to the intron-exon junction, we observed a homozygous10 bp deletion between positions -26 and -17 (c.2281-26_-17del). This deletion co-segregated with the disease in the family, and was not detected in public databases nor in 101 ethnically-matched control individuals. In silico analysis predicted that this deletion would lead to altered intron 21 splicing. Bioinformatic analysis predicted that a

©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected].

ARVO 2015 Annual Meeting Abstracts recognition site for the SRSF2 splicing factor is located within the deleted sequence. Conclusions: Here we report a novel and unique intronic mutation of PROM1, underlying autosomal recessive CRD in a consanguineous Israeli family. Altered splicing probably results from deletion of a recognition site for the SRSF2 splicing factor. This report expands the spectrum of pathogenic mutations of PROM1 and further demonstrates the importance of intronic mutations. Commercial Relationships: Tamar Ben-Yosef, None; Osnat Eidinger, None; Rina Leibu, None; Hadas Newman, None; Leah Rizel, None; Ido Perlman, None Support: Foundation Fighting Blindness Program Number: 2899 Poster Board Number: C0155 Presentation Time: 8:30 AM–10:15 AM Novel Genotype-Phenotype correlations in a Ma– ori and Polynesian Population with Autosomal Recessive Inherited Retinal Disease, using a Next Generation Targeted Retinal Panel. Andrea L. Vincent1, 2, Nandoun Abeysekera1, Katherine van Bysterveldt1, Verity F. Oliver1, Graeme C. Black3, 4. 1Ophthalmology, University of Auckland, Auckland, New Zealand; 2Eye, Greenlane Clinical Centre, Auckland, New Zealand; 3Manchester Centre for Genomic Medicine, Institute for Human Development, University of Manchesster, Manchester, United Kingdom; 4Central Manchester University Hospitals NHS Foundation Trust, Manchester Academic Health Sciences Centre (MAHSC), St Mary’s Hospital, Manchester, United Kingdom. Purpose: To phenotypically characterize presumed autosomal recessive rod-cone retinal dystrophies (ARRP) in Ma–ori and Polynesian patients with no previous identified gene mutation; to elucidate the gene mutations which are associated with retinal dystrophies in this population, and to establish phenotype-genotype correlations. Methods: Thirty-six patients of Ma– ori and Polynesian ancestry with no causative mutation detected on disease microarrays (Asper, Estonia), were identified from the Inherited Retinal and Optic Nerve Disorders Database. Clinical history and examination, pedigrees, OCT, fundal photography, fundus autofluorescence, and electrophysiology were obtained and reviewed for each patient. DNA of 15 of these patients with presumed ARRP underwent Next Generation Sequencing of a targeted retinal disease gene panel (NGSTRDGP). An ethnically matched control population was screened for identified changes. Subsequent patients seen with a similar phenotype were tested for the novel change. Results: Of the Ma– ori and Polynesian patients studied, probable disease-causing mutations were detected in 40% using NGS-TRDGP: this included a hemizygous RPGR mutation (n=1), heterozygous CRX (n=1), and a novel homozygous PDE6B mutation, predicted to be deleterious (n=4, all New Zealand Ma–ori). The phenotype in these patients is consistent with previously described PDE6B -associated disease. This change was present in 1/124 an ethnically matched alleles. Identification of 3 subsequent Ma– ori patients with a similar phenotype to that observed with PDE6B, has confirmed the same homozygous PDE6B variant. Conclusions: NGS is a superior technique to other commercially available techniques for causative gene mutations in ARRP. The majority of Ma– ori and Polynesian patients tested have no disease causing pathogenic variant(s) detected, which is strongly supportive of novel genetic mechanisms causative of disease in this population. The novel PDE6B mutation, is not in high frequency in a Ma–ori control population, and predicted to be deleterious. The phenotypegenotype correlation allows identification of the likely causative gene, resulting in targeted and cost effective gene screening. Within

the Ma– ori and Polynesian population there are still unknown genetic mechanisms accounting for the majority of disease. Commercial Relationships: Andrea L. Vincent, None; Nandoun Abeysekera, None; Katherine van Bysterveldt, None; Verity F. Oliver, None; Graeme C. Black, None Support: Retina New Zealand, Fight for Sight UK, Save Sight Society NZ. Program Number: 2900 Poster Board Number: C0156 Presentation Time: 8:30 AM–10:15 AM TSPAN12 mutation spectrum of a cohort of Familial Exudative Vitreoretinopathy patients Xianjun Zhu1, 2, Periasamy Sundaresan3, Yu Zhou1, 2, Xiong Zhu1. 1 Center for Human Molecular Genetics, Sichuan Provincial Hospital, Chengdu, China; 2School of Medicine, University of Electronic Science and Technology of China, Chengdu, China; 3Department of Genetics, Aravind Medical Research Foundation Aravind Eye Hospital, Madurai-625 020, India. Purpose: Familial exudative vitreoretinopathy (FEVR) is a group of inherited retina diseases characterized by defective retinal vessels. Tetraspanin 12 (TSPAN12) was identified as a cause of FEVR in several genetic studies. The purpose of this study was to identify novel TSPAN12 mutations in a cohort of India patients with FEVR and to describe the associated clinical phenotypes. Methods: Written consent was obtained form all subjects in this study. Clinical phenotypes of the patients with TSPAN12 mutations were documented.Whole exome sequencing was applied to a cohort of 27 families of FEVR patients. All variants were verified by Sanger sequencing. Effect of geteic variants on protein funcyion were pridicted by SIFT and Polyphen spoftware. Effects of idenfied mutations on TSPAN12 protein function were assayed for the Norrinβ-catenin signaling pathway with luciferase reporter assays. Results: Five novel heterozygous mutations in TSPAN12 were identified. All mutations involved highly conserved residues and were not present in 500 normal individuals. In SuperTopFlash (STF) cell line, four of these mutants failed to induce luciferase reporter activity in response to Norrin. Conclusions: Five novel TSPAN12 mutations in India patients with autosomal dominant FEVR were identified. Similar to that of FZD4 and LRP5, the phenotypes associated with TSPAN12 mutations showed variation in disease severity among members of the same family. Commercial Relationships: Xianjun Zhu, None; Periasamy Sundaresan, None; Yu Zhou, None; Xiong Zhu, None Support: NSFC 81271007, 81470668. Program Number: 2901 Poster Board Number: C0157 Presentation Time: 8:30 AM–10:15 AM Visual Outcomes of Japanese Patients with Retinitis Pigmentosa and Usher Syndrome Caused by USH2A Mutations Kimiko Suto1, Katsuhiro Hosono1, Yasunori Nagase1, Hiroshi Nakanishi2, Kunihiro Mizuta2, Shinsei Minoshima3, Yoshihiro Hotta1. 1 Ophthalmology, HAMAMATSU UNIVERSITY SCHOOL OF MEDICINE, Hamamatsu, Japan; 2Otorhinolaryngology/Head & Neck Surgery, Hamamatsu University School of Medicine, Hamamatsu, Japan; 3Photomedical Genomics, Hamamatsu University School of Medicine, Medical Photonics Research Center, Basic Medical Photonics Laboratory, Hamamatsu, Japan. Purpose: EYS and USH2A are the most common causative genes for retinitis pigmentosa (RP) in Japan. We analyzed 14 patients with two deleterious USH2A mutations to determine the clinical findings of Japanese patients with RP and Usher syndrome type II (USH2) associated with USH2A mutations. The visual field and visual acuity

©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected].

ARVO 2015 Annual Meeting Abstracts of patients were tested and compared with those previously reported in association with EYS mutations. Methods: Two nonsyndromic RP patients and 12 USH2 patients with previously identified USH2A mutations were included in this study. The complete history and medical records of the patients were reviewed, and clinical evaluations were performed using standard procedures, including spectral-domain optical coherence tomography (OCT). Electroretinography (ERG) was also performed for some patients. Otolaryngological examination, including audiography, was performed for all patients. Goldmann visual fields scanned with a Canon or Epson scanner were analyzed using ImageJ software, and the field areas of the V-4e targets were measured and compared with the normal areas. Results: There were six males and eight females with an age range of 12 to 52 years at the time of initial examination. 11 patients had a family history and 3 were isolated cases. All 14 patients had night blindness. In all patients, the fundus displayed changes typical of RP. OCT images also showed a marked decrease in the retinal thickness, resulting from the loss of photoreceptor layers. ERG responses were consistent with severe generalized rod–cone dysfunction. Overall, most patients showed relatively well-preserved visual acuity in their 30s, with rapid deterioration in their 40s or 50s. The constriction of visual fields was symmetric. Concentric constriction initiated in the 20s or 30s, and no effective residual visual field was observed after the 50s. These findings indicated that the visual outcome of RP in patients with USH2A mutations was consistent with that of RP associated with EYS mutations. Conclusions: RP in Japanese patients with USH2A mutations is characterized by rod–cone dystrophy and shows a poor prognosis after 60 years of age. Further studies with a larger sample size are needed to clarify these findings. Commercial Relationships: Kimiko Suto, None; Katsuhiro Hosono, None; Yasunori Nagase, None; Hiroshi Nakanishi, None; Kunihiro Mizuta, None; Shinsei Minoshima, None; Yoshihiro Hotta, None

©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected].