Program ISGEDR Ghent 2013 - · PDF fileHelen!DIMARAS! Toronto,!Canada! 15:36! Detection!of!calcifications!in!RB!using!gradientK echo!MRimaging!sequences:!comparative!

The Meeting of the International Society for Genetic Eye Diseases & Retinoblastoma

Ghent, Belgium

August 22 – 24, 2013

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Welcome to Ghent and to Your Meeting … Dear Participant Allow us to wish you a very warm welcome to the wonderful city of Ghent for the 2013 Meeting of the ISGEDR. With a program filled to the brim with fantastic presentations, we are confident that you will find this meeting most stimulating. The scientific quality of the papers and posters given by the participants covers a broad spectrum of genetic eye disease and retinoblastoma. Mixed with the free papers, are a number of symposia and keynote lectures. In addition to the superb intellectual gems of the free papers and posters, the symposia and keynote lectures will most certainly update us on the latest developments in therapies for inherited retinal dystrophies, current concepts of fibrillinopathies and related disorders, recognition and management of seeding of retinoblastoma, genome-‐wide association studies, and how novel molecular genetic techniques influence current ophthalmic genetic practice. We are certain that you may also find the beauty of Ghent and its many cultural highlights a treat. In addition to lunch provided on site, other culinary delights include a Belgian beer tasting with assorted cheeses on Thursday evening, and a gala dinner on Saturday evening in Hôtel Falligan, the most important rococo building of Ghent, built in 1755. We hope you have a great ISGEDR 2013 Meeting! Bart P. Leroy, M.D., Ph.D. Meeting Chair, and Head of Local Organizing Committee David A. Mackey, M.D. President, ISGEDR Elias I. Traboulsi, M.D. Executive Vice-‐President, ISGEDR Brenda Gallie, M.D. Member, Scientific Program Committee Birgit Lorenz, M.D. Member, Scientific Program Committee

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The International Society for Genetic Eye Diseases ISGEDR

Mission Statement

To bring together individuals interested in the field of genetic diseases of the eye and in retinoblastoma

*** To provide a forum for researchers in the field of genetic

diseases of the eye to share information ***

To promote international collaborations in the study of genetic diseases of the eye and in retinoblastoma

*** To disseminate scientific knowledge through international conferences

and through its official publication, Ophthalmic Genetics

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The International Society for Genetic Eye Disease & Retinoblastoma wishes to thank the following sponsors who have generously supported the 2013 ISGEDR Meeting

Platinum sponsor

Gold sponsors

Silver sponsor

Bronze sponsors

Sponsors

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A Special Thank You

To four people without whom the ISGEDR 2013 Meeting wouldn't be what it is:

Mrs. Sandy WONG, Cleveland, OH, USA Mrs. Ingrid VERVENNE, Ghent, BELGIUM

Mrs. Mayya TRABOULSI, Cleveland, OH, USA Mrs. Véronique LEROY-‐DERVEAUX, Ghent, BELGIUM

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Thursday August 22, 2013

9:00 -‐ 9:15 Welcome Dr. David MACKEY

Dr. Bart P. LEROY 9:15 -‐ 9:30 Official Opening Dr. Anne DE PAEPE

Geneticist & Rector-‐Elect Ghent University

9:30 -‐ 11:00 Genetics Session 1 Clinical Ophthalmic Genetics

Chairs: Birgit LORENZ & Elias I TRABOULSI

9:30 Autosomal recessive bestrophinopathy Rajani BATTU

Bangalore, India 9:42 Regulatory mutations in the 5'-‐UTR of

NMNAT1, cause Leber congenital amaurosis Frauke COPPIETERS, Ghent, Belgium

9:54 Adult-‐onset cone dystrophy associated with carboxyl RPGR mutations

Rola BA-‐ABBAD London, UK

10:06 Is the visual function seen in a child with lipemia retinalis reversible?

Hanna L SCANGA Pittsburgh, PA, USA

10:18 Benign yellow dot dystrophy Arundhati DEV BORMAN London, UK

10:30 Bestrophinopathy with low penetrance of BEST1 mutation?

Meghan MARINO Cleveland, USA

10:42 Characterization of photoreceptor structure in LCR-‐deletion associated blue cone monochromatism

Robert HUFNAGEL Seattle, WA, USA

10:54 Identification of concurrent PRPH2 & RP2 mutations within an apparent autosomal dominant pedigree: when are we done testing?

Dianne KH WHEATON Dallas, TX, USA

11:06 Copy number analysis of ABCA4 in Belgian patients with Stargardt disease reveals exon 20-‐22 deletion

Miriam BAUWENS Ghent, Belgium

11.18 The number of sequence variations detected in ABCA4 correlates with poorer VA in a cohort of Stargardt disease patients

Virginia UTZ Cleveland, OH, USA

11:20 -‐ 12:15 Break Poster Viewing

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12:15 -‐ 13:00 Franceschetti Lecture Dr. Edwin M STONE Iowa City, IA, USA Introduced by Dr. David MACKEY

13:00 -‐ 14:00 Lunch -‐ Provided on Site Poster Viewing

14:00 -‐ 15:48 Retinoblastoma Session 1 Diagnosis, Epidemiology & Genetics

Chairs: Brenda GALLIE & Ashwin REDDY

14:00 Retinoblastoma clinical research in Egypt: 5

years experience Ahmad SAMIR Cairo, Egypt

14:12 Squint as a presenting feature in retinoblastoma

Ashwin REDDY London, UK

14:24 Molecular surveillance for metastatic retinoblastoma

Brenda GALLIE Toronto, Canada

14:36 Second non-‐ocular tumors among survivors of retinoblastoma treated with proton radiotherapy

Shizuo MUKAI Boston, MA, USA

14:48 Diagnostic performance of MRI and CT for retinoblastoma: a systematic review and meta-‐analysis

Marcus DE JONG Amsterdam, The Netherlands

15:00 Delay in the diagnosis of retinoblastoma in the UK: has anything changed in 2 decades?

Ashwin REDDY London, UK

15:12 Outcomes of intra-‐ and extra-‐ocular retinoblastomas from a single institute in South India

Parag SHAH Tamil Nadu, India

15:24 Eye cancer pathology in Kenya Helen DIMARAS Toronto, Canada

15:36 Detection of calcifications in RB using gradient-‐echo MR imaging sequences: comparative study between in vivo MR imaging and ex vivo high-‐resolution CT

Pim DE GRAAF Amsterdam, The Netherlands


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16:30 -‐ 18:42 Genetics Session 2 Anterior Segment, Glaucoma, Ocular Development & Syndromes

Chairs: Petra Liskova & Elise HEON

16:30 ADAMTS9 as a candidate gene in pathogenesis

of anterior segment dysgenesis Johane DUBAIL Cleveland, OH, USA

16:42 Expansion of the ocular phenotype caused by mutations in ADAMTS18

Gavin ARNO London, UK

16:54 Epithelial recurrent erosion dystrophy: a misnomer?

Walter LISCH Hanau, Germany

17:06 Homozygous mutations in PXDN cause congenital cataract, corneal opacity and developmental glaucoma

Kamron KHAN Leeds, UK

17:18 Non-‐selective assembly of fibrillin microfibrils in the rodent ocular zonule and in vitro: implications for Marfan syndrome

Lauren C BEENE Cleveland, OH, USA

17:30 Ocular features of microcephaly with or without chorioretinopathy, lymphedema, or mental retardation (MCLMR) syndrome due to KIF1 mutations

Irina BALIKOVA Ghent, Belgium

17:42 Harboyan syndrome in a Czech proband caused by a novel homozygous nonsense mutation in SLC4A11

Petra LISKOVA Prague, Czech Republic

17:54 Brittle cornea syndrome: characterization of a retinal phenotype with relevance to common ocular disease

Louise PORTER Manchester, UK

18:06 Identity-‐by-‐descent mapping and exome sequencing reveals new candidate gene for primary congenital glaucoma locus GLC3E

Hannah VERDIN Ghent, Belgium

18:18 Eye developmental anomalies and the vitamin-‐A pathway

Nicky RAGGE Oxford, UK

18:30 MSX2 gene duplication with eye development defect

Julie PLAISANCIE Strasbourg, France

18:45 -‐ 20:45 Belgian Beer Tasting Het Pand

Please note that this may not be a complete meal -‐ See list of restaurants

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Friday August 23, 2013

08:30 -‐ 09:54 Genetics Session 3 Genetic Testing

Chairs: Frauke COPPIETERS & Lisa KEARNS

8:30 Search for New Genes in Autosomal Recessive

Retinitis Pigmentosa Nour-‐Al-‐Dain MARZOUKA Montpellier, France

8:42 Genetics of isolated unilateral retinoblastoma Dietmar LOHMANN Duisburg-‐Essen, Germany

8:54 A novel approach to molecular diagnosis of retinoblastoma using next-‐generation sequencing and RB1 custom ACGH

Gemma D'ELIA Rome, Italy

9:06 The Role of Genetic Testing in Elucidating the Cause of Early Onset Horizontal Nystagmus (EOHN)

Joanne SUTHERLAND Toronto, Canada

9:18 Losing the "middle man" in genetic eye disease: the current scope of direct to consumer testing in ophthalmology

Lisa S KEARNS Melbourne, Australia

9:30 Next-‐generation sequencing panel to determine the real prevalence for gene defects underlying rod-‐cone dystrophies

Said EL SHAMIEH Paris, France

09.42 Identification of novel homozygous deletions in consanguineous pedigrees as a shortcut to candidate gene discovery in inherited retinal dystrophies

Kristof VAN SCHIL Ghent, Belgium

09:55 -‐ 10:40 François Lecture: "Fibrillinopathies and related disorders -‐ Molecular considerations and ocular phenotypes"

Dr. Elias I TRABOULSI, Cleveland, OH, USA Introduced by Dr. Bart P. LEROY


11:15 -‐ 13:00 Symposium: Update on Gene Therapy & Stem Cell Therapy for Retinal Dystrophies

Chairs: Mark PENNESI & Bart P LEROY

11:15 How do you deliver gene therapy to the eye? Albert M MAGUIRE

Philadelphia, PA, USA

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11:30 Retinal dystrophies: defining new endpoints for therapeutic trials

Birgit LORENZ Giessen, Germany

11:45 Gene therapy for Leber congenital amaurosis Jean BENNETT Philadelphia, PA, USA

12:00 Gene therapy for choroideraemia Mandeep SINGH Oxford, UK

12:15 Gene therapy for Stargardt disease Isabelle AUDO Paris, France

12:30 Gene therapy for Usher syndrome type 1B Mark PENNESI Portland, OR, USA

12:45 Stem cell therapy for retinal dystrophies Mandeep SINGH Oxford, UK


13:45 -‐ 14:15 Business Meeting of ISGEDR All Members David MACKEY Presiding

14:15 -‐ 15:00 Genetics Session 4 Therapy for retinal dystrophies

Chairs: Arlene DRACK & John G FLANNERY

14:15 AAV-‐RDCVF rescues cones and AAV-‐RDCVFL

protects rods in retinal degeneration John G FLANNERY Berkeley, CA, USA

14:45 Immunosuppressive therapy for retinal degeneration in Batten disease

Arlene DRACK Iowa City, IA, USA

15:00 -‐ 16:00 Symposium Genome Wide Association Studies (GWAS)

Chairs: Christopher HAMMOND & David MACKEY

15:00 Ophthalmic success with Genome Wide

Association Studies (GWAS) David MACKEY Perth, Australia

15:15 An international GWAS of Glaucoma-‐Related Optic Disc Parameters: the International Glaucoma Genetics Consortium

Henriët SPRINGELKAMP Rotterdam, The Netherlands

15:30 GWAS of refractive error Christopher HAMMOND London, UK

15:45 GWAS of central corneal thickness and keratoconus

Seyhan YAZAR Perth, Australia

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16.00 -‐ 16.30 Break Poster Viewing

16.30 -‐ 17:54 Genetics Session 5 Multifactorial Genetics & Behavioural Studies


16:30 Role of genetic loci implicated in diabetic

retinopathy risk Annie MCAULEY Melbourne, Australia

16:42 No evidence for differential IL17RC promoter methylation in age-‐related macular degeneration

Maria FRANCHINA Perth, Australia

16:54 Association of genetic risk for refractive error and time spent outdoors

Seyhan YAZAR Perth, Australia

17:06 Enrichment of pathogenic alleles in the brittle cornea gene ZNF469 provides novel insights into development of corneal thinning & keratoconus

Louise PORTER Manchester, UK

17:18 Description of intellectual, adaptive & behavioral function of patients with Bardet-‐Biedl syndrome: preliminary report

Elise HEON Toronto, Canada

17:30 Reproductive behavior of individuals with increased risk of having a child

Charlotte DOMMERING Amsterdam, The Netherlands

17:42 -‐ 19.15 Poster session All authors required to stand by poster

19:15 -‐ bedtime

Free Evening in Ghent See list of restaurants

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Saturday August 24, 2013

8:30 -‐ 09.54 Retinoblastoma Session 2 Treatment

Chairs: Ahmad SAMIR & Annette Moll

8:30 Management of bilateral retinoblastoma with

chemo reduction and focal therapy Natalia MATTI Tijuana, Mexico

8:42 Focal treatment of retinoblastoma tumors with simultaneous 810nm and 532nm lasers

Ashwin MALLIPATNA Bangalore, India

8:54 Standard conservative approaches using systemic CT and local treatments: experience of the Institut Curie

Isabelle AERTS Paris, France

9:06 Management and outcome of 550 cases of retinoblastoma treated at a tertiary referral center in India

Bhavna CHAWLA New Delhi, India

9:18 EBR therapy for retinoblastoma resistant to chemotherapy and focal therapy: outcome and predictive factors -‐ CANCELLED

Yacoub A YOUSEF Amman, Jordan

9:30 Trilateral retinoblastoma at the age of nine weeks

Vicktoria VISHNEVSKIA-‐DAI. Tel Aviv, Israel

9:42 Conservative treatment of advanced bilateral retinoblastoma: comparison of 1995-‐2003 to 2004-‐2009

Livia LUMBROSO-‐LE ROUIC -‐ Paris, France

09:55 -‐ 10:40 Ellsworth Lecture: "Seeding in retinoblastoma: recognition, management, and regression patterns"

Dr Francis MUNIER, Lausanne, Switzerland-‐ Introduced by Dr Brenda GALLIE


11:30 -‐ 12:54 Retinoblastoma Session 3 Treatment & Miscellaneous

Chairs: Francis MUNIER & Hossam EL-‐ZOMOR

11:30 Intravitreal melphalan for retinoblastoma with

cloudy vitreous seeds Marie-‐Claire GAILLARD, Lausanne, Switzerland

11:42 Intra-‐arterial chemotherapy using multi-‐agent chemotherapy for treatment of recalcitrant

Brian MARR New York, NY, USA

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intra-‐ocular retinoblastoma 11:54 High-‐risk histopathologic features of

retinoblastoma in Egypt: treatment outcomes Hossam EL-‐ZOMOR Cairo, Egypt

12:06 Pathological findings of retinoblastoma in Egypt; implementing CAP protocol in developing countries

Hala TAHA Cairo, Egypt

12:18 Sodium iodide symporter is differentially expressed in retinoblastoma and correlates with the aggressiveness of tumors

Jaisy Samuel Tamil Nadu, India

12:30 Identification of most commonly used web-‐based search terms for leukocoria

Sandra E STAFFIERI Crawley, Australia

12:42 High-‐resolution SNP array profiling identifies variability in retinoblastoma genome stability

Berber MOL


14:00 -‐ 14:54 Genetics Session 6 Clinical & Diagnostic Aspects

Chairs: Birgit LORENZ & Arif O KHAN

14:00 Tuberous sclerosis -‐ why we need an

ophthalmic check-‐up Christina GERTH-‐KAHLERT -‐ Zürich, Switzerland

14:12 Color vision in Stargardt disease Tine VANDENBROUCKE, Ghent, Belgium

14:24 The phenotypic variability of retinal dystrophies associated with mutations in CRX

Sarah HULL London, UK

14:36 Pathognomonic retinal dysfunction and dystrophies in children

Arif O KHAN Riyadh, Saudi Arabia

14:48 Phenotype of RDH12-‐related early-‐onset retinal dystrophy

Julie DE ZAEYTIJD Ghent, Belgium

15:00 -‐ 16:30 Symposium

“How will increasingly efficient genetic diagnostics impact your patients?”

• What needs to happen to take advantage of genetics?

• How are you using genetics in patient care now, and in 3 years?

• How will you determine the most cost-‐effective approach (timing, genes tested, clinical practice)?

Chairs: Elise HEON & Edwin M STONE

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Each speaker will address these questions in the context of their clinical expertise, followed by a panel discussion with opportunity for all to explore issues and opportunities of rapidly evolving new technologies.

14:55 Introduction to symposium + my experience,

views, ideas & queries Elise HEON, Toronto, Canada

15:00 My experience, views, ideas & queries Isabelle AUDO, Paris, France

15:05 My experience, views, ideas & queries Andrew WEBSTER/Anthony MOORE, London, UK

15:10 My experience, views, ideas & queries Arlene DRACK, Iowa City, IA, USA

15:15 My experience, views, ideas & queries Helen DIMARAS, Toronto, Canada

15:20 My experience, views, ideas & queries Bart P LEROY, Ghent, Belgium & Philadelphia, PA, USA

15:25 Panel Discussion 16:10 Questions Panelists

16:25 Chairperson Summary Elise HEON, Toronto,

Canada 16:30 -‐ 17:00 Break

Poster Viewing

**** PLEASE NOTE THAT THE FOLLOWING SESSION IS INTENDED FOR PATIENTS & THEIR FAMILIES

****

17:00 -‐ 18:30 Belgian RP Patients Session Association Retina Pigmentosa -‐ Ophthalmic Genetics

Chairs: Viviane HALLET-‐TORDEURS & Bart P LEROY

17:00 Introduction Bart P LEROY

Ghent, Belgium & Philadelphia, PA, USA

17:05 Basics of Genetics Elias I TRABOULSI Cleveland, OH, USA

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17:25 Update on Genetic Testing Edwin M STONE Iowa City, IA, USA

17:45 Using Genetic Tests in the Clinic Elise HEON Toronto, Canada

18:05 Update on Gene Therapy Jean BENNETT Philadelphia, PA, USA

18:25 Conclusions Bart P LEROY, Ghent Belgium & Philadelphia, PA, USA

19:30 -‐ ON Gala Dinner All Registered Participants

Hotel Falligan

Kouter, 172, 9000 Ghent

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Thursday August 22, 2013

9:00 -‐ 9:15 Welcome Dr. David MACKEY

Dr. Bart P. LEROY 9:15 -‐ 9:30 Official Opening Dr. Anne DE PAEPE

Geneticist & Rector-‐Elect Ghent University

9:30 -‐ 11:00 Genetics Session 1 Clinical Ophthalmic Genetics

Chairs: Birgit LORENZ & Elias I TRABOULSI

9:30 -‐ AUTOSOMAL RECESSIVE BESTROPHINOPATHY. RAJANI BATTU; SATHI DEVI; PADMAMALINI MAHENDRADAS; RAJESH S KUMAR; ROHIT SHETTY. Bangalore, India Introduction: Autosomal recessive Bestrophinopathy (ARB) is a recessively inherited retinal disorder characterized by multiple punctate yellowish lesions, subretinal fluid, subretinal exudates, hyperopia and narrow angles; most patients present with central vision loss or night blindness. ARB is the result of a complete absence of functional bestrophin-‐1 protein in the RPE. All ARB patients carry a BEST1 mutation on both chromosomes. We reviewed the literature relevant to ARB and present a series of patients who presented with clinical features of Bestrophinopathy Materials & Methods: Objective: To describe the clinical and electrophysiological features of patients with autosomal recessive Bestrophinopathy. Design: Retrospective case series. Methods: All five patients underwent a complete ophthalmic examination including a fundus photo and Spectral-‐domain optical coherence tomography (OCT). Electroretinogram (ERG) and Electro-‐oculogram (EOG) were done in four patients. Main outcome measures: Age at onset, presence of shallow anterior chamber, refractive error, visual acuity, fundus appearance, features on fundus autofluorescence (FAF), OCT, ERG and EOG. Results: The age of onset varied from 8 years to 49 years. Fundus abnormalities included multiple vitelliform lesions, subretinal deposits and subretinal scars. Four of the five patients had intraretinal or subretinal fluid. Two out of the five patients had angle closure glaucoma and needed bilateral trabeculectomies; one patient needed bilateral peripheral iridotomies. The refractive error was variable; none of them were significantly hyperopic. Three patients showed abnormal scotopic and photopic responses on the full field ERG while one patient showed abnormal scotopic responses only. All four patients who underwent the EOG had a reduced Arden ratio (<1.5). One patient did not undergo the electrophysiology tests. Conclusions: ARB is a clinically recognizable phenotype with distinguishing features on the FAF, OCT and ERG. Considering the increased risk these patients have for angle-‐closure glaucoma, either a prophylactic or therapeutic YAG laser PI and a close monitoring of the intraocular pressures may be essential. It is also necessary to screen

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the family at risk for narrow angles and glaucoma. Although not essential for diagnosis, analysis of the BEST1 gene may be useful for genetic counseling and prognostication. 9:42 -‐ REGULATORY MUTATIONS IN THE 5'-‐UTR OF NMNAT1, ENCODING THE NUCLEAR ISOFORM OF NICOTINAMIDE NUCLEOTIDE ADENYLYLTRANSFERASE 1, CAUSE LEBER CONGENITAL AMAUROSIS. FRAUKE COPPIETERS, ANNE LAURE TODESCHINI, FRANCOISE MEIRE, ELFRIDE DE BAERE, BART P. LEROY, Ghent, Belgium Introduction: Leber Congenital Amaurosis (LCA) is the earliest inherited retinal dystrophy (RD). Recently, coding mutations in NMNAT1 resolved the last remaining locus, LCA9 (Falk et al. 2012). Here, we aimed to identify the genetic defect in an LCA9-‐linked consanguineous Sub-‐Saharan African family (F1) and determine the contribution of NMNAT1 mutations in LCA. Materials & Methods: The proband of F1 underwent massive parallel sequencing (MPS) of all exons and promoter regions located in the 4 largest IBD regions (NimbleGen Sequence Capture 385K array, Roche GS FLX Titanium). NMNAT1 Sanger sequencing was performed on gDNA (exons) and cDNA. NMNAT1 expression analysis was performed on leukocyte cDNA with qPCR. F1 and F2 underwent thorough phenotyping. Luciferase assays were performed using the pGL3 Luciferase Reporter Vectors and SwitchGear Genomics promoter constructs. Results: MPS identified in F1 a novel homozygous 5â?TUTR variant in NMNAT1 (c.-‐70A>T), which segregated with disease. Subsequent NMNAT1 Sanger sequencing in 76 probands with LCA/early-‐onset RD revealed coding mutations in 5 additional probands, and identified a second homozygous 5â?TUTR variant (c.-‐69C>T) in a Moroccan consanguineous LCA family (F2). In both F1 and F2, cDNA sequencing detected no other potential pathogenic variants but revealed loss of heterozygosity in heterozygous carriers, suggesting NMNAT1 mRNA degradation. Subsequently, significantly lower mRNA expression in leukocytes was shown for the homozygous mutants from F1 and F2 in comparison with healthy controls. Luciferase assays for both adjacent 5â?TUTR variants in RPE-‐1 cells revealed significantly lower activity for both variants in comparison with the wild-‐type 5â?TUTR. Of note, both families show LCA with evolutive macular involvement, typical for NMNAT1-‐related disease. Conclusions: In conclusion, this study sustained the role of coding NMNAT1 mutations in LCA. Moreover, the identification of two neighboring 5â?TUTR variants in NMNAT1 makes this the first study to link 5â?TUTR regulatory mutations to congenital blindness, and may impact upon the role of 5â?TUTR mutations in hereditary blindness in general. 9:54 -‐ ADULT-‐ONSET CONE DYSTROPHY ASSOCIATED WITH CARBOXYL RPGR MUTATIONS. ROLA BA-‐ABBAD; ANTHONY G ROBSON; PANAGIOTIS SERGOUNIOTIS; VINCENT PLAGNOL; ANDREW R WEBSTER, Moorfields Eye Hospital & UCL Institute of Ophthalmology, London, UK Introduction: Retinal dystrophy associated with RPGR mutations usually manifest in males as severe, early-‐onset dystrophy. This paper describes the molecular pathology

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and clinical phenotype in two unrelated simplex cases of adult-‐onset cone dystrophy associated with mutations in RPGR exon 15 (ORF15). Materials & Methods: Thirty probands presenting with macular or cone dystrophy, atypical and/or negative for ABCA4 retinopathy, and consistent with recessive inheritance were each analyzed using exome-‐sequencing. Clinical assessment included ophthalmic examination, fundus autofluorescence (FAF), ocular coherence tomography (OCT) and electroretinography (PERG; ERG). Results: Each of two simplex male patients was found to be hemizygous for a different frame shifting mutation in the 3’ end of ORF15: c.3178_3179delAG, p.E1060fs; and c.3317dupA, p.K1106fs, respectively. Both patients became symptomatic during the 4th decade and complained of glare and poor color discrimination. The visual acuity at presentation was in the range of 6/9-‐6/18, and at the last follow up visit was 6/15-‐6/18. Both were unable to read standard color vision testing plates. The patients had moderate or high myopia, with a bilateral spherical correction of -‐5.50 D, and -‐10 D, respectively. Only mild pigment changes were evident on ophthalmoscopy. The FAF and OCT showed evidence of RPE and photoreceptor outer-‐segment disruption at the macula, the peripheral retina appearing normal. Pattern ERGs were consistent with severe macular dysfunction in both cases. The first patient had ERG evidence of severe progressive generalized cone system dysfunction. The second patient showed ERG evidence of mild generalized cone system loss that was stable over 2 years. Clinical examination of each obligate carrier showed normal findings at age 64 and 77 years, respectively. Conclusions: Although male patients with retinal dystrophy associated with RPGR mutations usually manifest with a severe, early-‐onset dystrophy, those with mutations close to the 3’ end of ORF15 can present with a much less severe adult-‐onset cone dystrophy. This may suggest a preferential effect of RPGR-‐ORF15 hypomorphic alleles on foveal cones. Such cases may be good candidates for gene-‐replacement therapy as early intervention may arrest progression before the onset of significant visual loss. Female carriers may not manifest retinal disease. 10:06 -‐ IS THE VISUAL DYSFUNCTION SEEN IN A CHILD WITH LIPEMIA RETINALIS REVERSIBLE? HANNAH L. SCANGA; VALERIA FU; KEN K. NISCHAL. Pittsburgh, PA, USA Introduction: Lipemia retinalis is described as a rare and asymptomatic condition that may occur in patients with extreme elevations in plasma triglyceride levels.(1) Here, we report decreased visual acuity and retinal dysfunction secondary to lipemia retinalis in a young patient with Familial Lipoprotein Lipase (LPL) deficiency, with subsequent improvement as a result of strict dietary control. Materials & Methods: Retrospective case review and serial fundus photography of a single female patient referred for ophthalmology examination. Results: A 4-‐year-‐old girl of consanguineous parents presented for ophthalmology evaluation. She was known to have molecularly confirmed LPL deficiency, hypotonia, and developmental delay. Examination, including fundus photography, revealed decreased visual acuity, strabismus, and lipemia retinalis. ERG showed an abnormal rod-‐cone driven response. At the time of evaluation, VLDL cholesterol levels were 847

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mg/dl and triglyceride levels were 4,235 mg/dl. After 3 months of strict dietary control, photoreceptor response and retinal appearance normalized, and visual acuity improved. Conclusions: Retinal dysfunction has been previously demonstrated by electrophysiology in patients: with lipemia retinalis, but visual acuity was not impaired.(2) In our case, we speculate that decreased visual acuity could be secondary to sustained high levels of cholesterol and triglycerides due to LPL deficiency. The exact mechanism of the visual dysfunction other than a mechanical effect is unclear. Ultimately, strict dietary control can reverse any visual dysfunction and normalize the retinal appearance. References: 1. Diwan AG, Dabadghao VS, Gupta A. Lipemia retinalis. JAPI 2011; 59: 650. 2. Chao-‐Kung L, Shih-‐Jen C, Dau-‐Ming N, Chia-‐Chen T, Fenq-‐Lih L, Wen-‐Ming H. Electrophysiologic changes in lipemia retinalis. Am J of Ophth 2005; 139:1142-‐5. 10:18 -‐ BENIGN YELLOW DOT DYSTROPHY. ARUNDHATI DEV BORMAN, MARTINA SUZANI, TONY MOORE. London, UK Introduction: Considerable clinical and genetic heterogeneity exists for the rare group of disorders that encompass the inherited macular dystrophies. We present a new macular phenotype, which may occur in isolation or as a familial trait that is associated with excellent visual function. Materials & Methods: This was a retrospective observational case series. All patients underwent complete ocular examinations. Fundus autofluorescence (FAF) imaging, spectral domain optical coherence tomography (sdOCT) and electrophysiological studies were performed where possible. Results: Nineteen affected individuals were identified from 15 unrelated families. In 2 families the condition segregated in an autosomal dominant manner. The condition was sporadic in 13 individuals. The median presenting age was 15 years (range 4-‐45 years). Thirteen of 19 subjects were female. The majority of subjects were asymptomatic, either presenting after a routine optometry visit or following screening due to a positive family history. The 7 symptomatic subjects presented with reduced vision. In 4/7 the reduced vision was attributed to uncorrected hypermetropia, with 2/4 being amblyopic. General health was normal in all but 2 patients: one had depression; another had suffered a cerebrovascular accident during treatment for acute lymphoblastic leukaemia. Eleven of 19 subjects had normal visual acuity. Colour vision was normal. There was no associated refractive error in those with normal vision. All subjects had multiple, bilateral, symmetric yellow dots at the macula. In 11 subjects these were more numerous in the nasal parafoveal region. The dots appeared discrete in 14 individuals but more confluent in 5. In one subject, dots were also visible outside the vascular arcades in the right eye, scattered throughout the posterior pole. In 17 subjects a yellow crescent was visible, to a variable degree, around the optic disc, which was otherwise normal in all. In some individuals this appeared as a “yellow halo” around the disc; in others the change was more consistent with peri-‐papillary atrophy. The retinal periphery and vasculature were normal in all subjects. The dots were hyperfluorescent on autofluorescence imaging.

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sdOCT imaging demonstrated subtle irregularities in the region between the inner segment / outer segment junction and the retinal pigment epithelium. Full-‐field and pattern ERGs were normal. Conclusions: Although there are some similarities to the grade 1 lesions seen in North Carolina Macular Dystrophy, we describe a predominantly sporadic condition that represents a novel clinical entity that is associated with excellent visual function. 10:30 -‐ CLINICAL FINDINGS IN ACHROMATOPSIA WITH PDEC6 MUTATIONS. MARINO MJ, TRABOULSI EI. Cleveland Clinic, Cleveland, OH, USA. Purpose:To describe the clinical and OCT findings in a patient with ACHM and PDEC6 mutations Methods: The proband was evaluated in clinic. Family and medical histories were reviewed. Fundus photographs, fundus autofluorescence, and OCT were obtained. Molecular genetics testing was obtained. Results: Mutations were found in PDEC6. Fundus examination was normal except for blunting of the foveal reflex. FAF showed hypoAF in the center of the fovea and OCT showed a pathognomonic subfoveal cystic space. Conclusions: Mutations in PDEC6 cause less than 2% of cases of achromatopsia. OCT reveals a lesion pathognomonic for achromatopsia and guides the molecular diagnosis. 10:42 -‐ CHARACTERIZATION OF PHOTORECEPTOR STRUCTURE IN LCR-‐DELETION ASSOCIATED BLUE CONE MONOCHROMATISM. ROBERT HUFNAGEL; ROBERT A SISK; MICHEL MICHAELIDES; MAUREEN NEITZ; JOSEPH CARROLL; ZUBAIR M. AHMED, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA Introduction: Blue cone monochromatism (BCM) is an X-‐linked disorder of cone-‐driven vision caused by defective expression or function of both OPN1LW (encoding red opsin) and OPN1MW (encoding green opsin). Complete loss of red and green opsins occurs with deletion of the Locus Control Region (LCR), a critical enhancer necessary for expression of both genes. Patients with BCM typically have reduced visual acuity, diminished color discrimination, photophobia, and nystagmus. While BCM does not usually demonstrate progressive visual acuity loss or retinal deterioration, little is known about the effects of opsin loss on photoreceptor structure and function. Additionally, the fovea is comprised predominantly of red and green cone photoreceptor outer segments, indicating that BCM is an important model for foveal disease. Here, we describe genotype-‐phenotype analyses of four families segregating BCM. Materials & Methods: DNA was purified from blood samples obtained from affected individuals, parents, and unaffected siblings. Genomic segments harboring the LCR, promoter sequences, and OPN1LW and OPN1MW exons were analyzed by PCR amplification followed by direct Sanger sequencing, and gene arrangement and copy number were evaluated by RT-‐PCR. Clinical diagnosis included clinical assessment, color vision testing, family history, and fullfield and S-‐cone electroretinography (ERG). Macular and photoreceptor structure was evaluated by Optical Coherence Tomography (OCT) and Adaptive Optics (AO) imaging.

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Results: All four BCM families harbor deletions of the enhancer LCR and, therefore, lack expression of OPN1MW and OPN1LW genes. Probands ranged in age from 8 to 35 years old. Affected individuals had moderate impairment in best-‐corrected visual acuity, impaired cone ERGs, and reduced central foveal thickness with retinal pigmented epithelium (RPE) mottling. AO imaging of foveal photoreceptors indicated changes to foveal photoreceptor outer segment structure. Conclusions: These families with LCR-‐deletion associated BCM have similar phenotypic manifestations, including thin foveae with granular RPE, correlating with outer segment structural changes. Disease severity did not correlate with age or visual acuity. Ongoing quantification of these changes in patients with LCR-‐deletion associated BCM is necessary to better understand whether these are common among BCM patients and to identify optimal therapeutic strategies for this disease. 10:54 -‐ Identification of Concurrent PRPH2 and RP2 Mutations Within an Apparent Autosomal Dominant Retinitis Pigmentosa (adRP) Pedigree: When Are We Done Testing? DIANNA K.H. WHEATON, DAVID G. BIRCH, KAYLIE D. WEBB, STEPHEN P. DAIGER. University of Texas Health Science Center Houston, TX Introduction: Identify the genetic cause of RP in a 5-‐generation, apparent autosomal dominant pedigree with multiple variably affected male and female family members. Materials & Methods: The female proband (4th generation; age 24) presented for visual function [e.g., electroretinogram (ERG), visual fields, retinal imaging] and genetic assessments. Family medical history was obtained emphasizing ophthalmic conditions. Blood for genetics was submitted to a CLIA-‐certified lab and analyzed using standard dideoxy fluorescent sequencing. Four additional family members presented later for testing. Results: The pedigree depicted affected individuals (n=5; 3M/2F) in 4 maternally-‐related generations, although descent to the proband appeared to involve reduced penetrance in her mother. Initial impressions suggested autosomal dominant although X-‐linked inheritance could not be excluded due to absent male-‐to-‐male transmission. Proband’s vision assessment indicated notable interocular differences. OD measures: rod ERG amplitude reduced 66%, cone flicker amplitude reduced 74%, and fields showed general reduced sensitivity. OS measures: rod ERG amplitude non-‐detectable, cone flicker reduced 95%, and field constricted to <10°. Subsequent testing of proband’s grandmother and mother found both to be variably affected. Grandmother (65yrs) exhibited normal rod ERG function, normal-‐to-‐borderline reduced cone flicker, and reduced peripheral field sensitivity; mother (46yrs) exhibited an intermediate phenotype with 65% rod reduction, normal cone flicker, and borderline abnormal fields. Proband’s genetic testing identified a Ser289Leu mutation in PRPH2 previously described in cone dystrophy. Analysis of the grandmother’s sample later identified an RP2 mutation; the Lys260X mutation is novel, however numerous similar disease-‐causing nonsense mutations have been reported in RP2 making this likely pathogenic. Segregation analysis for the identified mutations among family members found 3 of 3 RP affected relatives harbored the RP2 mutation. The proband harbored both

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mutations, inheriting the originally identified PRPH2 mutation from her undiagnosed father; her son (5th generation) was later found to have RP due to the RP2 mutation. Conclusions: Families rarely present en masse for clinical workups. Family history contracts/expands with inclusion of additional historians leading to evolution of the pedigree; phenotypic characterization also evolves. When has enough data been collected? Perhaps, when a disease-‐causing mutation consistent with the phenotype is identified? When the mutation is confirmed in additional family member(s)? Such concerns have strong implications for genetic counseling and directing genetic testing. In this case, further testing identified the predominant cause of disease in the family and also identified a possible explanation for increased severity of RP in the proband, namely concurrent RP2 and PRPH2 mutations. 11:06 -‐ COPY NUMBER ANALYSIS OF ABCA4 IN BELGIAN PATIENTS WITH STARGARDT REVEALS EXON 20-‐22 DELETION. MIRIAM BAUWENS, HANNAH VERDIN; THOMY DE RAVEL DE L’ARGENTIERE; ELFRIEDE DE BAERE; FRAUKE COPPIERTERS; Center for Medical Genetics Ghent, Ghent University Hospital, Ghent, Belgium Introduction: Stargardt disease (STGD1) is one of the most frequent autosomal recessive retinal dystrophies, with a prevalence of 1/8000. Genetic testing for STGD1 is currently mostly limited to Sanger sequencing of the coding region of ABCA4. This approach leaves up to 30% of the STGD1 patients without a complete molecular diagnosis. In order to identify a second mutation, we performed quantitative PCR (qPCR) to screen for copy number variations (CNVs) in patients with one heterozygous or no ABCA4 mutation. Materials & Methods: CNV screening comprised 50 qPCR assays, performed in 48 patients with one heterozygous or no mutation in ABCA4 following ABCA4 Asper chip and/or Sanger sequencing, and 27 controls (LC480, Roche). Breakpoint delineation was performed by additional qPCR assays, by sequencing using internal sequencing primers as well as next-‐generation sequencing (NGS) technology (Illumina, Miseq). Results: CNV analysis revealed a heterozygous deletion of exon 20-‐22 in a family segregating both STGD1 disease and autosomal recessive retinitis pigmentosa. The index patient and her affected brother were heterozygous for the splice site mutation c.5461-‐10C>T and this deletion. Using additional qPCR assays, followed by sequencing with internal sequencing primers and NGS of a patient specific junction PCR product, we could delineate the breakpoint regions to a total of 100 nucleotides. This deletion covers 4 kb, spans exons 20-‐22 and probably corresponds with a previously identified deletion of exons 20-‐22 in ABCA4 (Maugeri et al. 1999). Analysis of the putative breakpoint regions reveals the presence of Alu elements, suggesting nonallelic homologous recombination as a possible mechanism underlying this deletion. Additional patients with STGD1 or allied disorders with only one or no identified coding ABCA4 mutation are being screened for this specific deletion using junction PCR. Haplotype analysis will be performed in additional deletion patients. Conclusions: qPCR screening of the ABCA4 coding region identified a deletion of exon 20-‐22.To our knowledge only three genomic rearrangements of ABCA4 have been

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described so far (Rozet et al. 1999, Maugeri et al. 1999, Yatsenko et al. 2003). Our and previous studies using high-‐resolution CNV analysis such as multiplex ligation-‐dependent probe amplification (MLPA) or qPCR suggest a very low prevalence of ABCA4 deletions in STGD1, assuming the occurrence of non-‐coding variations or complex rearrangements in the remaining 30% of patients with only one or no coding mutation. 11:18 -‐ THE NUMBER OF SEQUENCE VARIATIONS DETECTED IN ABCA4 CORRELATES WITH POORER VISUAL ACUITY IN A COHORT OF PATIENTS WITH STARGARDT DISEASE. V. MIRALDI-‐UTZ; R. COUSSA; M. MARINO; A. CHAPPELOW; G. PAUER; S. HAGSTROM; E.I. TRABOULSI. Cole Eye Institute, Cleveland, OH and Abrahamson Eye Institute, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA Purpose: To assess the genotypic diversity in patients with Stargardt disease (STGD); to characterize the time course of vision loss and clinical progression; and to determine genotype-‐phenotype correlates. Methods: Retrospective, cross-‐sectional and observational study where the history, examination, and molecular diagnostic results of 112 consecutive patients with a clinical diagnosis of STGD were reviewed. The relationship between age of presentation and last follow-‐up visit, best-‐corrected visual acuity (BCVA) in the better-‐seeing eye, and ABCA4 genotypic findings were evaluated by cross-‐sectional and/or longitudinal analyses. The effect of age groupings on BCVA was evaluated using ANOVA analyses. An alpha error of 0.05 was deemed statistically significant. Age-‐normalized visual acuity was compared between patients with 0 to 1 disease-‐associated ABCA4 sequence variations versus 2 or more for all methods of genetic testing utilized, (n=112) and those in whom gene sequencing of all 50 exons was performed (n=29). Results: The average age at initial consultation was 29.8+15.7 years (range, 6-‐78 years). Of the 112 patients, 98 patients carried a probable molecular diagnosis of STGD, based upon the presence of at least one disease-‐associated sequence variant in the ABCA4 gene. Cross-‐sectional linear and polynomial regression analyses of age at presentation versus visual acuity were not significant (p=0.35 and p=0.82 respectively). In regard to genotype-‐phenotype correlates, patients who harbored 2 or more disease-‐associated sequence variants had worse age-‐normalized BCVA than those with 0 (p=0.01) or 1 variant (p=1.27x10-‐7) identified. Likewise, for those whose ABCA4 gene was fully sequenced, those with 2 or more variants had worse age-‐normalized visual acuity than those with 1 variant (p=0.0008). Patients (n=16) with c.5882G>A allele (p.G1961E) demonstrated better age-‐normalized BCVA than the remaining population (p=0.01). In contrast, patients (n=7) with a c.5461-‐10T>C variation demonstrated early disease onset and macular pigmentary clumping consistent with advanced disease in older patients. Conclusions: Presenting visual acuity does not follow a linear or polynomial distribution with respect to age. Those who present in the first decade have worse visual acuity on presentation than those who present in subsequent decades, and those with worse BCVA at initial presentation have a steeper rate of visual decline. Those with 2 or more disease-‐associated sequence variations have worse BCVA than those in whom no or only 1 identifiable variation is present. Select sequence variations in ABCA4 may confer a

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specific phenotype. This information may assist in counseling patients and determining visual prognosis and candidacy for treatment. 11:20 -‐ 12:15

Break Poster Viewing

12:15 -‐ 13:00

Franceschetti Lecture Dr. Edwin M STONE Iowa City, IA, USA Introduced by Dr. David MACKEY

13:00 -‐ 14:00

Lunch -‐ Provided on Site Poster Viewing

14:00 -‐ 15:48

Retinoblastoma Session 1 Diagnosis, Epidemiology & Genetics

Chairs: Brenda GALLIE & Ashwin REDDY

14:00 -‐ RETINOBLASTOMA CLINICAL RESEARCH IN EGYPT: 5 YEARS EXPERIENCE. AHMAD SAMIR ALFAAR, RADWA NOUR, MOHAMED KAMAL, MOHAMED SABRY BAKRY, SAMEERA EZZAT, SHERIF ABOUELNAGA; Children's Cancer Hospital -‐ Egypt (ISGEDR TRAVEL AWARD RECIPIENT) Introduction and purpose: Institution-‐lead clinical research is a systematic effort for understanding diseases and its management in relation to local situation and needs. In Egypt, Children’s Cancer Hospital Egypt has initiated a clinical research program that aims at standardizing treatment protocols for different diseases including Retinoblastoma. The aim of this study is to present the steps we’ve conducted, specific challenges we’ve faced, key performance indicators that we’ve designed and achievements we have accomplished during the development of our Retinoblastoma clinical research program in Egypt. Methods: During the evolution of this program we’ve tracked events and progress in the retinoblastoma program over an online shared application. This was reported on observational basis by research team for practices of clinical, pharmacy, nursing and other supporting teams beside research team itself. Retinoblastoma Research Team has used shared online documents for facilitating collaborative building of reports. Results: Between July 2007 and July 2012 we’ve adopted 5 treatment protocols. We’ve enrolled 250 patients over those protocols. Follow-‐up of challenges revealed different factors related to nature of the retinoblastoma disease and management, knowledge of clinical and research teams, settings in developing countries and factors related to registration and follow-‐up of huge numbers of patients. Physicians were engaged in the

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program through weekly discussions and tumour board meetings. We’ve implemented a tailored training program for nurses and pharmacists about the disease and treatment protocols. The research team has developed an online protocols portal to disseminate the latest versions of treatment roadmaps to clinical management teams. We succeeded to integrate a real-‐time follow-‐up system that reports full picture of retinoblastoma patients’ performance. Significance: This model represents an example for facilitating the transformation into clinical research in developing countries with the aid of international best practices and information technology tools. The paper represents a blueprint for other institutions in low resource countries to integrate its unique knowledge about the disease and its response into practice. 14:12 -‐ REPRODUCTIVE BEHAVIOR OF INDIVIDUALS WITH INCREASED RISK OF HAVING A CHILD. CHARLOTTE DOMMERING; MIRJAM GARVELINK; HANNE MEIJERS-‐HEIJBOER; LIDEWIJ HENNEMAN; ANNETTE MOLL; JENNIFER VAN DIJK; SASKIA IMHOF; VU University Medical Center, Amsterdam, The Netherlands Purpose: To investigate reproductive decisions of individuals at risk for a child with retinoblastoma (Rb), and examine factors influencing these decisions. Materials & Methods: Cross-‐sectional questionnaire survey, 1-‐10 years post-‐genetic counseling of individuals with an increased risk of a child with Rb who visited the National Retinoblastoma Treatment Center in the Netherlands. Risk for having a child with Rb ranged from <1% to 50%. Results: The response rate was 69% (81/118). Of 43 respondents who considered having children after becoming aware of their increased risk, 25 (58%) indicated that Rb influenced their decisions, of whom 14 had a recurrence risk of less than 3%. Twenty respondents decided against having more children and 5 used prenatal diagnosis. Eighteen of the 43 respondents had children (or more children) and did not use any of the alternative reproductive options, although half indicated having had doubts about their decisions. Only perceived risk was significantly associated with Rb influencing reproductive behavior by multiple logistic regression (P = 0.003). Of 17 respondents planning children (or more children), 11 (65%) considered using one of the alternative reproductive options in the future. Conclusions: Reproductive behavior is greatly influenced by Rb and perceived risk, not objective risk, is the most important factor of influence. It is important to offer individuals at increased risk continued access to genetic counseling, even when this risk is small. 14:24 -‐ MOLECULAR SURVEILLANCE FOR METASTATIC RETINOBLASTOMA. BRENDA L. GALLIE, ELISE HEON, DIANE RUSHLOW, DONCO MATEVSKI, HELEN DIMARAS, ROSEANNE SUPERSTEIN, PATRICK HAMEL, WILLIAM HALLIDAY, FRANCOIS CODERE, ANNE-‐SOPHIE CARRET, BENJAMINE ELLEZAM, HELEN SL CHAN, Sick Kids Hospital and University of Toronto, Toronto, Canada Introduction: High sensitivity detection of extraocular retinoblastoma tumor cells provides an opportunity to diagnose and treat metastatic retinoblastoma as early,

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increasing the potential for cure. We compared conventional cytology to molecular assay, using the identified primary tumor gene mutations as personal biomarkers for minimal residual disease. Materials & Methods: We conducted a retrospective chart review of patients for whom both conventional and molecular surveillance for metastatic disease had been performed. Results: Surveillance for metastatic cells was performed for two reasons: i) poor prognosis pathology in an enucleated eye, when we treat with 4-‐cycle chemotherapy and harvest stem cells prophylactically (7 patients); or ii) metastatic eye or trilateral retinoblastoma, when we treat with chemotherapy and harvested stem cells for transplant, after screening a stem cell aliquot for tumor cells (4 patients). For patients with a germline RB1 mutation (M1), their tumor-‐specific RB1 mutations (M2 or M3 to Mn) were unique identifiers. For patients with non-‐germline RB1 mutations (M1), M1 or M2 were unique identifiers. Sensitivity for cells carrying mutations detectable by optimized PCR was higher than cytology, while one copy number mutation was detectable at equivalent sensitivity. Conclusions: Early diagnosis of metastasis or recurrence can support early intervention, which is anticipated to increase the chance for cure. 14:36 -‐ SECOND NON-‐OCULAR TUMORS AMONG SURVIVORS OF RETINOBLASTOMA TREATED WITH PROTON RADIOTHERAPY. SHIZUO MUKAI; ROSHAN SETHI; DAVID Y. KIM; JOHN E. MUNZENRIDER; SHANNON M. MACDONALD, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, MA, USA Introduction: Mortality for patients with hereditary retinoblastoma is usually from second non-‐ocular malignancy. Since radiotherapy increases this risk, it is avoided despite its high rate of tumor control. Proton radiotherapy can significantly spare non-‐target tissue. We reviewed the risk of both in-‐field and out-‐of-‐field second malignancy for the largest and oldest cohort of retinoblastoma patients treated with proton therapy. Materials & Methods: Retrospective review of all patients with hereditary retinoblastoma treated with proton therapy at the Massachusetts General Hospital between 1986 and 2011. Results: 54 patients were identified. Median follow-‐up from the start of radiotherapy was 6.9 years (range, 11.3 months to 24.4 years). The median age at follow-‐up was 9.0 years (range, 31.3 months to 24.5 years). 15 patients had more than 10 years of follow-‐up from the start of radiotherapy with 20 who were older than 10 years at last follow-‐up. Total person-‐years of follow-‐up was 417.2. We identified one secondary malignancy, a non-‐metastatic osteosarcoma of the distal femur outside of the radiation field that developed 9.0 years after the initiation of radiotherapy. No second malignancy was seen within the radiation field of any patient. The cumulative incidence of all second tumors in the proton cohort was 5% at 10 years, but 0% for in-‐field malignancy. Conclusions: In the largest and oldest cohort of retinoblastoma patients treated with proton therapy, no second malignancy was seen in the field of radiotherapy. While

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longer follow-‐up is necessary, our data suggest that the risk of radiation-‐associated malignancy is minimal with proton therapy. 14:48 -‐ DIAGNOSTIC PERFORMANCE OF MAGNETIC RESONANCE IMAGING AND COMPUTED TOMOGRAPHY FOR RETINOBLASTOMA: A SYSTEMATIC REVIEW AND META-‐ANALYSIS. MARCUS DE JONG; PIM DE GRAAF; DANIEL P. NOIJ; ANNETTE C. MOLL; JONAS A. CASTELIJNS, VU University Medical Center, DeBoelelaan, Amsterdam Introduction: To determine and compare the diagnostic performance of magnetic resonance imaging (MRI) and computed tomography (CT) for the diagnosis of tumor-‐extent in retinoblastoma, using histopathology as reference standard. Materials & Methods: Medline and Embase were searched for literature published until April 2013 assessing the diagnostic performance of MRI and/or CT in detecting intra-‐ and extraorbital tumor extension of retinoblastoma. Diagnostic accuracy data were extracted from included studies. Summary estimates were based on a random-‐effects model. Intra-‐ and inter-‐study heterogeneity was analyzed. Results: Data of the following tumor-‐extent parameters were extracted: anterior eye segment involvement, ciliary body, optic nerve, choroidal, and (extra-‐)scleral invasion. Articles on MRI reported results of 591 eyes from 14 studies and CT yielded 257 eyes from 4 studies. For post-‐ laminar optic nerve, choroidal, and scleral invasion conventional MRI showed sensitivities of 59% (95%CI: 37â?”78%), 74% (95%CI: 52â?”88%), and 89% (95%CI: 19â?”100%) respectively and specificities of 94% (95%CI: 84â?” 98%), 72% (95%CI: 31â?”94%), and 99% (95%CI: 81â?”100%) respectively. MRI with a high (versus a low) image quality showed higher diagnostic accuracies for detection of post-‐laminar optic nerve and choroidal invasion, but these differences were statistically not significant. Studies reporting on the diagnostic accuracy of CT did not provide enough data to perform any meta-‐analyses. Conclusions: MRI is an essential diagnostic tool for the detection of local tumor-‐ extent in retinoblastoma although its diagnostic accuracy, especially sensitivity, might be improved with higher spatial resolution. The benefit of CT over MRI cannot be demonstrated and this technique, as it uses ionizing radiation, should be avoided in this highly sensitive population. 15:00 -‐ DELAY IN THE DIAGNOSIS OF RETINOBLASTOMA IN THE UK: HAS ANYTHING CHANGED IN 2 DECADES? M. ASHWIN REDDY; MARCUS POSNER; ADIL JAULIM; MANDEEP S. SAGOO, Moorfields Eye Hospital NHS Foundation Trust, London, UK Introduction: To re-‐audit the child with retinoblastoma’s journey from primary care to a specialist Retinoblastoma unit. An audit conducted from 1993 to 1996 demonstrated a median referral time from primary to tertiary care of 56 days. The median referral time for patients requiring post-‐enucleation chemotherapy showed significant delays of 189 days. Materials & Methods: Retrospective audit using patient notes from January 2006 to December 2009. Only sporadic cases were included.

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Results: Of 37 patients, median referral time from primary to secondary health care was 12 days with transition from the primary to tertiary service of 13.5 days. Median time intervals were 7 days for GPs from primary to secondary care. Twelve patients required post-‐enucleation chemotherapy for local invasion. There was no significant delay between this group and those who did not require post-‐enucleation chemotherapy (p>0.05). Conclusions: Median time to diagnosis compared to the 1990s has reduced significantly from 56 to 13.5 days. However, referral times between healthcare professions still vary from 4 to 127 days and lengthy delays should be avoided. Notably the patient journey for children who require post-‐enucleation chemotherapy is not significantly delayed suggesting that delays in diagnosis may not be the only factor determining morbidity in aggressive invasive retinoblastoma. 15:12 -‐ OUTCOMES OF INTRA AND EXTRA OCULAR RETINOBLASTOMAS FROM A SINGLE INSTITUTE IN SOUTH INDIA. PARAG SHAH; NARENDRAN VENKATAPATHY; KALPANA NARENDRAN; Aravind Eye Hospital, Tamilnadu State, India Introduction: To study the outcome of intra and extra ocular retinoblastomas from a single institute in South India. Materials & Methods: Retrospective interventional case series involving 105 cases diagnosed to have retinoblastoma from January 2006 till December 2011. Standard treatment modalities ranging from focal therapy for group A, systemic chemotherapy along with focal therapy for group B, C and D were given. Additionally injections of subtenon carboplatin were given to 14 eyes with group C/D disease. External beam radiotherapy was given only if vitreous seeds recurred or in cases of orbital spread and was given only when the child was more than 1 year of age. Iodine 125 brachytherapy was given to 3 eyes for tumor recurrence. Results: The mean age of presentation was 20.1 months (range 5 days â?” 120 months). There were 67 unilateral and 38 bilateral cases. Leucokoria was the commonest presentation seen in 79% (83 cases). 112 of 141 eyes presented with advanced disease (group D/E). Globe salvage rates were 100% for group A (11 eyes), B (16 eyes) and C (2 eyes). For group D, eye salvage rate was 29.5% (10/34 eyes), however 8/34 eyes were primarily enucleated. There were 9 cases with orbital spread (stage 3) of which 5 (55.5%) survived at a mean follow up of 33.6 months (range 21-‐48 months). All these cases received 6 cycles of chemotherapy, enucleation and 46 Gy of external beam radiation. At the overall mean follow up of 36 months the survival rate was 90.5% with 10 deaths (9.5%). The commonest cause of death (7/10) was distant metastasis due to refusal to take initial treatment. Two died because of chemo complications and one because of congenital heart disease. Conclusions: Globe salvage and survival rates from India are getting closer to the rates seen in developed nations. The commonest cause of death seen our study was distant metastasis secondary to refusal for initial treatment.

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15:24 -‐ EYE CANCER PATHOLOGY IN KENYA. HELEN DIMARAS; ELIZABETH DIMBA; WAIRIMU WAWERU; JESSIE, GITHANGA; KAHAKI KIMANI, University of Toronto, Toronto, Canada Introduction: We are leading a Kenyan initiative to determine the impact of digital pathology on the care of retinoblastoma. Materials & Methods: We developed a centralized lab in Nairobi that receives retinoblastoma specimens from all over Kenya and processes them using approved standard operating procedures. Images are produced by a digital scanner, and pathology results are disseminated and discussed online in real-‐time. Results: Accurate and timely pathology resulted in enhanced care of Kenyan retinoblastoma patients. Integration of digital technology to support pathology services supported knowledge transfer and skills transfer. A bidirectional educational network of local pathologists and other clinicians in the circle of care of the patients emerged and served to emphasize the clinical importance of cancer pathology at multiple levels of care. A robinhood business model of healthcare service delivery was developed to support sustainability and scale-‐up of cancer pathology services. Conclusions: When embraced as an integrated component of cancer care in Kenya, digital pathology enhances care of current patients, and offers the opportunity for frequent connection and consultation for development of expertise over time. Sustaining Kenyan capacity in cancer pathology may be facilitated by combining key principles of science & technology, social science and business. 15:36 -‐ DETECTION OF CALCIFICATIONS IN RETINOBLASTOMA USING GRADIENT-‐ECHO MR IMAGING SEQUENCES: COMPARATIVE STUDY BETWEEN IN-‐VIVO MR IMAGING AND EX-‐VIVO HIGH RESOLUTION CT. PIM DE GRAAF; FIRAZIA RODJAN; PAUL VAN DER VALK; THEODORA HADJISTILIANOU; ALFONSO CERASE; PAOLO TOTI; MARCUS C. DE JONG; ANNETTE C. MOLL; JONAS A. CASTELIJNS; PAOLO GALLUZZI; ON BEHALF OF THE EUROPEAN RETINOBLASTOMA IMAGING COLLABORATION (ERIC), VU University Medical Center, Amsterdam, The Netherlands Introduction: Intratumoral calcifications are very important in the diagnosis of retinoblastoma. Although CT is considered to be superior in detecting calcification, the radiation hazard in especially hereditary retinoblastoma patients should be avoided. The purpose of our study is to validate the value of T2*WI for detection of calcifications in retinoblastoma with ex-‐vivo CT as gold standard. Materials & Methods: Twenty-‐two consecutive patients with retinoblastoma (mean age, 21 months, range 1-‐71 months) with enucleation as primary treatment were imaged with a 1.5 T using a dedicated surface coil. Signal intensity void (SIV) indicating calcification on T2*WI were compared with ex-‐vivo high resolution CT (HRCT) and correlation was scored by two independent observers as poor, moderate or good correlation. Other parameters included shape and location of SIVs. In five tumors susceptibility-‐weighted images (SWI) were evaluated. Results: All calcifications visible on HRCT could be matched with SIVs on T2*WI and correlation was scored as good in 17 (77%) and moderate in 5 (23%) eyes. In total, 93% (25/27) of the SIVs inside the tumor correlated with calcifications compared to none

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(0/6) of the SIVs outside the tumor. Areas of nodular shaped SIVs correlated with calcifications in 92% (24/26) and linear shaped SIVs correlated with hemorrhage in 67% (6/9) of the cases. The correlation between SIVs on SWI was better in 4 out of 5 tumors compared to T2*WI. Conclusions: The results of our study show a good correlation between SIVs as detected on in-‐vivo T2*-‐weighted or SWI MR imaging sequences and calcifications on ex-‐vivo HRCT in retinoblastoma. Gradient-‐echo sequences may be helpful in suggesting the diagnosis of retinoblastoma. In retinoblastoma patients it is important to always avoid radiation hazards, especially from CT, and therefore the combination of fundoscopy, ultrasound and high-‐resolution MR imaging with gradient-‐echo sequences should become the standard diagnostic approach to diagnose retinoblastoma 15:48 – THE CASE FOR UPDATING THE INTERNATIONAL INTRAOCULAR RETINOBLASTOMA CLASSIFICATION. A. LINN MURPHREE, Los Angeles, CA, USA 16:00 -‐ 16:30 Break

Poster Viewing

16:30 -‐ 18:42 Genetics Session 2 Anterior Segment, Glaucoma, Ocular Development & Syndromes

Chairs: Eduardo SILVA & Elise HEON

16:30 -‐ ADAMTS9 AS CANDIDATE GENE IN PATHOGENESIS OF ANTERIOR SEGMENT DYSGENESIS. JOHANE DUBAIL; STEPHANIE HAGSTROM; ELIAS I. TRABOULSI; MEGHAN MARINO; GAYLE PAUER; SUNEEL APTE, Cleveland Clinic Lerner Research Institute, Cleveland, OH, USA Introduction: Anterior Segment Dysgenesis (ASD) comprises a genetically and phenotypically heterogenous group of congenital disorders resulting from anomalous development of ocular anterior segment structures and is an important cause of severe visual impairment in infants and young adults. Although mutations in a limited number of genes, including FOXC1, PITX2, PITX3, FOXE3, PAX6 and CYP1B1, were identified in ASD in humans, they do not explain the etiology of all cases and the pathogenesis of ASD is still not completely understood. ADAMTS9 is the most conserved member of the ADAMTS protease family, comprising secreted metalloproteases having multiple thrombospondin type I repeats, which are post-‐translationally modified by O-‐fucosylation. Adamts9-‐/-‐ mice die prior to initiation of embryonic eye development. Our studies demonstrated that Adamts9+/-‐ mice developed a fully penetrant congenital ASD whose most obvious sequelae were corneal opacity and neovascularization. The goal of this study is thus to understand how ADAMTS9 contributes to the formation of anterior segment structures such as cornea, iris, lens, ciliary body and aqueous drainage structures during eye development.

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Materials & Methods: Evolution of ASD and its consequences in Adamts9+/-‐ mice was defined by histological analysis of the eye during embryogenesis, post-‐natal growth and maturity and comparison to wild-‐type littermate eyes. Exon-‐by-‐exons equencing of ADAMTS9 was performed in selected patients presenting with ASD for which mutations in known causative genes were excluded. Results: Corneal opacity was observed inAdamts9+/-‐ mice as soon as their eyelids opened. In many mice, ocular enlargement occurred prior to or concurrently with corneal opacity, and in older mice, the affected eyes were greatly reduced in size, suggesting glaucoma followed by phtisis bulbi. Histological analyses of newborn Adamts9+/-‐ mouse eyes demonstrate the presence of various abnormalities in anterior segment structures. Particularly, a persistent adhesion between lens and cornea was visualized, identifying a Peters anomaly in newborn Adamst9+/-‐ mice. Those observations link this new mouse phenotype to human Peters-‐Plus Syndrome (PPS) resulting from defects in the enzyme B3GALTLthat is involved in O-‐fucosylation of TSRs. For these reasons, ADAMTS9 sequencing in ASD patients was undertaken, but no mutations have hitherto been identified. Conclusions: Collectively, those results and connection to PPS and O-‐fucosylation implicate ADAMTS9 in normal ocular anterior segment formation in mouse and render ADAMTS9 a strong candidate gene for Peters anomaly, PPS variants and/or others forms of ASD in humans. 16:42 -‐ EXPANSION OF THE OCULAR PHENOTYPIC CAUSED BY MUTATIONS IN ADAMTS18. GAVIN ARNO; AMAN CHANDRA; BIRGIT LORENZ; DAVID R FITZPATRICK; ANTHONY T MOORE, UCL Institute of Ophthalmology, London, UK Introduction: Homozygous mutations in ADAMTS18 have been described in 2 patients to date. We describe further patients and report the detailed ocular phenotype associated with mutations in this gene. Materials & Methods: Four affected patients from three families with an unusual ocular phenotype had full ophthalmic and systemic examination. Causative mutations were identified using homozygosity mapping with Sanger sequencing and massively-‐parallel sequencing of the whole exome. Results: All affected members from the three families had bilateral microcornea and a cone-‐rod dystrophy. In addition, a single affected individual in the first family had ectopic pupils, two affected brothers in a second family had childhood cataract, ectopia lentis and rhegmatogenous retinal detachment, a single affected individual in the third family had childhood cataract, smooth irides and rhegmatogenous retinal detachment. No occipital defects were seen in any of the four patients. Novel homozygous mutations in ADAMTS18 were identified in the first proband (c.1067T>A [p.L356*]), in the two affected brothers (c.2159G>C [p.C720S]) and in the third proband (c.1952G>A [p.R651Q]). All three mutations are predicted to be pathogenic. Conclusions: We confirm that recessive mutations in ADAMTS18 are associated with a range of ocular abnormalities, and provide further evidence that ADAMTS18 plays a key role in ocular development.

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16:54 -‐ EPITHELIAL RECURRENT EROSION DYSTROPHY: A MISNOMER ? WALTER LISCH, Johannes Gutenberg University Mainz, Hanau, Hessen, Germany Introduction: ERED represents one template of the IC3D-‐classification, published in 2008.To correct the term ERED due to new examinations. Materials & Methods: Clinical, histological, immunohistochemical, and genetical presentation of Franceschetti corneal dystrophy (FRCD). Results: Affected members of FRCD show in the first three decades only recurrent epithelial erosions and a complete recovery of the cornea. Diffuse, subepithelial corneal opacities develop in affected members in their fourth decade of life and older. Conclusions: FRCD, a new defined corneal dystrophy, is to differentiate against the Dystrophia Smolandiensis by today. The term ERED is a misnomer. Corneal dystrophies should be subdivided into two distinct groups regarding recurrent erosions of the corneal epithelium. Reference: Lisch W, Bron AJ, Munier FL, et al. Am J Ophthalmol 2012;153:1073-‐1081. 17:06 -‐ HOMOZYGOUS MUTATIONS IN PXDN CAUSE CONGENITAL CATARACT, CORNEAL OPACITY, AND DEVELOPMENTAL GLAUCOMA. KAMRON KHAN, King Khaled Eye Specialist Hospital, Saudi Arabia Anterior segment dysgenesis describes a group of heterogeneous developmental disorders that affect the anterior chamber of the eye and are associated with an increased risk of glaucoma. Here, we report homozygous mutations in peroxidasin (PXDN) in two consanguineous Pakistani families with congenital cataract-‐microcornea with mild to moderate corneal opacity and in a consanguineous Cambodian family with developmental glaucoma and severe corneal opacification. These results highlight the diverse ocular phenotypes caused by PXDN mutations, which are likely due to differences in genetic background and environmental factors. Peroxidasin is an extracellular matrix-‐associated protein with peroxidase catalytic activity, and we confirmed localization of the protein to the cornea and lens epithelial layers. Our findings imply that peroxidasin is essential for normal development of the anterior chamber of the eye, where it may have a structural role in supporting cornea and lens architecture as well as an enzymatic role as an antioxidant enzyme in protecting the lens, trabecular meshwork, and cornea against oxidative damage. 17:18 -‐ NON-‐SELECTIVE ASSEMBLY OF FIBRILLIN MICROFIBRILS IN THE RODENT OCULAR ZONULE AND IN VITRO: IMPLICATIONS FOR MARFAN SYNDROME. LAUREN C. BEENE; LAUREN W. WANG; DIRK HUBMACHER; DOUGLAS R. KEENE; DIETER P. REINHARDT; ROBERT P. MECHAM; ELIAS I. TRABOULSI; SUNEEL S. APTE. Department of Biomedical Engineering, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH, USA Introduction: Fibrillins, large secreted glycoproteins, are the major constituent of tissue microfibrils. FBN1 mutations leading to Marfan syndrome lead to deficiency of the microfibril-‐ composed ciliary zonule and lens subluxation (ectopia lentis). Here, analysis of Fbn1 knockout and mutant mouse eyes unexpectedly demonstrated an intact, and the authors examined its composition. Concordantly, the authors investigated the

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biogenesis of microfibrils in wild-‐type, fibrillin-‐1 and fibrillin-‐2 deficient fibroblast cultures. Materials & Methods: The ciliary zonules of wild-‐type, Fbn1 and Fbn2 knockout or mutant mice, as well as other animal species were evaluated by immunofluorescence using monospecific fibrillin-‐1 and fibrillin-‐2 antibodies. Antibody specificity was demonstrated by staining Fbn1 knockout and Fbn2 knockout tissues as controls. The zonule in Fbn1 knockout mouse eyes was examined by transmission electron microscopy. Microfibril biogenesis in wild-‐type, Fbn1 knockout and Fbn2 knockout mouse fibroblasts was evaluated by immunofluorescence. Results: Contrary to expectation, a zonule was histologically and ultrastructurally evident in Fbn1 knockout mouse eyes. Immunofluorescence demonstrated that both wild type and Fbn1 knockout mouse zonule, as well as rat and hamster zonule contained fibrillin-‐2, contrasting with the mature human and bovine zonule, which did not. Analysis of microfibril biogenesis in fibroblasts demonstrated that when both fibrillin-‐1 and fibrillin-‐2 were present, heterotypic microfibrils were readily formed; moreover, fibrillin-‐2 microfibrils formed readily in Fbn1 knockout cultures, and vice versa. Fibrillin-‐2 assembly in wild-‐type and Fbn1 knockout fibroblasts and human non-‐pigmented ciliary epithelial cells was fibronectin-‐dependent and initiated by cell surface punctate deposits which progressively elongated to form microfibrils. Conclusions: These studies suggest that microfibril composition depends substantially on the local levels of fibrillin isoform expression and that the assembly mechanism of fibroblasts is not highly selective in this regard. The assembly of fibrillin-‐2 microfibrils is dependent on fibronectin assembly in a manner similar to that previously shown for fibrillin-‐1. This raises the intriguing possibility that zonules could be generated or maintained in Marfan syndrome by inducing expression of fibrillin-‐2 in the ciliary body. The presence of a zonule comprising fibrillin-‐2 in the mouse and rat and an intact zonule in Fbn1 knockout mice limits the utility of these rodent models for studying ectopia lentis Marfan syndrome. 17:30 -‐ OCULAR FEATURES OF MICROCEPHALY WITH OR WITHOUT CHORIORETINOPATHY, LYMPHEDEMA, OR MENTAL RETARDATION (MCLMR) SYNDROME DUE TO KIF11 MUTATIONS. IRINA BALIKOVA; ANTHONY ROBSON; GRAHAM HOLDER; PIA OSTERGAARD; ANTHONY MOORE, Free University of Brussels, Brussels, Belgium Introduction: Microcephaly with or without chorioretinopathy, lymphedema, or mental retardation (MCLMR, OMIM 152950) is an autosomal dominant disorder characterized by primary microcephaly, growth retardation, developmental delay, lymphedema and chorioretinal dysplasia. The gene causing this disorder KIF11 has recently been identified (Ostergaard, 2012). KIF11 mutations occur in ~75% of patients clinically diagnosed with MCLMR. The associated chorioretinopathy is reported to occur in approximately 2/3 of mutation positive families (Ostergaard, 2012). Materials & Methods: Clinical assessment included best-‐corrected visual acuity (BCVA), anterior segment and dilated fundus examination, color fundus photography, fundus autofluorescence (FAF) and Spectral Domain (SD) OCT were recorded. Full-‐field and

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Pattern electroretinograms (ERGs) incorporated the International Society for Clinical Electrophysiology of Vision (ISCEV) standards and were performed with corneal (cases 1 and 4) or peri-‐orbital skin electrodes (cases 5 and 6). Cases 1 and 4 underwent additional On-‐Off-‐ ERGs. Results: Areas of chorioretinal atrophy outside the arcades were present in all patients except patient 5. Patient 7 had a retinal fold in the left eye and a right retinal detachment. ERGs showed mild-‐moderate generalized rod and cone photoreceptor dysfunction in 13 of 14 eyes. ERGs in the final eye were severely abnormal in keeping with the retinal detachment, (case 7). Additional inner retinal dysfunction was present in 3 cases (including 2 siblings) with cone On-‐pathway involvement (cases 1 and 4). Conclusions: Microcephaly may be associated with three different retinal phenotypes -‐ the typical microcephaly chorioretinopathy; microcephaly and a progressive rod-‐cone dystrophy, (Cantu, 1977;Atchaneeyasakul, 1998)or microcephaly and retinal folds (Jarmas, 1981;Angle, 1994). One subject in the present study has the typical chorioretinal atrophy in one eye and a retinal fold in the other. Similarly two siblings, presenting with the typical chorioretinal atrophy in the sister and retinal fold in the brother were reported (Trzupek, 2007). This data shows that some cases of microcephaly and retinal folds may have KIF11 mutations and this suggests that the underlying KIF11 mutation may affect both retinal and choroidal vascular development. 17:42 -‐ HARBOYAN SYNDROME IN A CZECH PROBAND CAUSED BY A NOVEL HOMOZYGOUS NONSENSE MUTATION IN SLC4A11. PETRA LISKOVA; LUBICA DUDAKOVA; ALISON J. HARDCASTLE, First Faculty of Medicine, Charles University in Prague, Czech Republic Introduction: The aim of this study was to identify the molecular genetic cause of Harboyan syndrome in a patient of Czech origin. Materials & Methods: Ocular examination and medical documentation analysis of a case from a non-‐consanguineous Caucasian family diagnosed with congenital hereditary endothelial dystrophy and deafness followed for more than two decades. DNA was collected and direct sequencing of all 19 exons of the SLC4A11 gene was performed. Results: The 55 year old female underwent iridectomy at the age of 3 months because of an initial diagnosis of juvenile glaucoma. During the course of her life three penetrating keratoplasties were performed in the right eye and two in the left eye, the first one at the age of 12 years. Upon the last examination the probandâ?Ts best corrected visual acuity was 0.05 in the right eye and hand movement with accurate light projection in the left eye. Audiological testing confirmed bilateral perceptive hearing impairment. A novel homozygous sequence variant c.2188C>T; p.(Arg730*) was identified in SLC4A11. The nature of the change, as well as absence in SNP and variant databases, supports its pathogenic nature. Conclusions: Identification of a novel mutation responsible for Harboyan syndrome in exon16 of SLC4A11 further confirms that the protein regions encoded by exons 16-‐18 are also important for the function of inner ear cells.

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17:54 -‐ BRITTLE CORNEA SYNDROME: CHARACTERISATION OF A RETINAL PHENOTYPE WITH RELEVANCE TO COMMON OCULAR DISEASE. LOUISE PORTER; FORBES MANSON; RICHARD BONSHEK; GRAEME BLACK; TARIQ ASLAM, University of Manchester and Manchester Royal Eye Hospital, UK Introduction: Brittle cornea syndrome (BCS) is a rare autosomal recessive disease characterised by extreme corneal thinning and rupture. Mutations in ZNF469 and PRDM5 cause BCS. How mutations in these genes cause BCS is not known, but both genes are reported to act on a common pathway regulating extracellular matrix components. We report clinical cases of a retinal phenotype in BCS and are investigating the role of PRDM5 as an epigenetic modifier involved in the development and maintenance of the retinal vasculature and Bruchâ?Ts membrane. Materials & Methods: Clinical case reports with advanced retinal imaging. Immunohistochemistry and gene expression profiling on ocular samples and cultured fibroblasts of BCS patients with PRDM5 mutations. Results: One patient with PRDM5 p. E134X mutation developed choroidal neovascularization bilaterally aged 26. Another patient with a PRDM5 R590X mutation had peripheral retinal vascular abnormalities detected upon ultra-‐widefield Optos imaging. Immunohistochemistry on the eyes of two individuals with a PRDM5 internal deletion of exons 9-‐14 revealed retinal thinning, retinal capillary paucity and absence of staining of Bruch’s membrane for collagens I and IV in the affected individuals versus a control sample. Conclusions: Results suggest a role for PRDM5 as an epigenetic modifier gene involved in the development and maintenance of the retina and its vasculature. The observation of choroidal neovascularization and retinal degeneration in BCS patients resembling age-‐related macular degeneration highlights a potentially novel extra-‐cellular matrix-‐related disease pathway with possible relevance to common retinal degenerative diseases. 18:06 -‐ IDENTITY-‐BY-‐DESCENT MAPPING AND EXOME SEQUENCING REVEALS A NEW CANDIDATE GENE FOR THE PRIMARY CONGENITAL GLAUCOMA LOCUS GLC3E. VERDIN HANNAH; BART LEROY; FRAUKE COPPIETERS; PHILIPPE KESTELYN; ELFRIDE DE BAERE, Ghent University, Ghent, Belgium Introduction: Primary congenital glaucoma (PCG) is caused by developmental anomalies of the trabecular meshwork and the anterior chamber angle, resulting in increased ocular pressure and optic nerve damage from early life. PCG mostly displays an autosomal recessive inheritance. To date, four PCG loci are known (GLC3A-‐D), with two genes identified, CYP1B1 and LTBP2. Here, we aimed to map the disease gene in a four-‐generation consanguineous family with PCG from Jordan. Materials & Methods: Mutations in known PCG genes were excluded. Identity-‐by-‐descent (IBD) mapping was performed in six affected members using genomewide SNP genotyping (250K, Affymetrix). Two affected individuals underwent exome sequencing (TruSeq Exome Enrichment; HiSeq, Illumina; CLC Bio). Results: IBD mapping revealed a common region of 11.5 Mb on 19p13.2-‐p13.11, being a new candidate PCG locus named GLC3E. Using IBD filtering of exome data and Ingenuity

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Variant analysis, we found a homozygous missense variant c.304C>T (p.R102C) in a novel candidate gene for GLC3E. This variant is predicted to be deleterious. It was absent in 718 Caucasian control chromosomes and was found in one out of 156 Jordan control chromosomes, being consistent with its reported minor allele frequency. The potential GLC3E might play a role in Toll-‐ and BMP-‐signalling pathways. In addition, its mutations might dysregulate mitochondrial complex I. Its expression domain in the eye anterior segment is currently being investigated in zebrafish and mouse. Conclusions: We identified a new candidate gene for the GLC3E locus, confirming the genetic heterogeneity of PCG, and possibly representing the third PCG gene. 18:18 -‐ EYE DEVELOPMENTAL ANOMALIES AND THE VITAMIN A PATHWAY. NICOLA RAGGE; NICOLAS CHASSAING; ARIANA KARIMENEJAD: PATRICK CALVAS; LUCAS FARES-‐TAIE; SYLVIE GERBER; JOSSELINE KAPLAN; JEAN-‐MICHEL ROZET, Oxford Brookes University, Oxford and Birmingham Women's Hospital, West Midlands, UK Introduction: Anophthalmia or microphthalmia (A/M) are severe ocular developmental anomalies, together responsible for up to 25% childhood blindness. So far, a genetic cause can be identified in 25%-‐40% of patients. Here we describe two A/M conditions involving the retinoic acid pathway: (1) PDAC (Pulmonary hypoplasia/agenesis, Diaphragmatic hernia,Anophthalmia/microphthalmia (A/M) and Cardiac Defect) or Matthew Wood syndrome, caused by recessive mutations in STRA6, the receptor protein STimulated by Retinoic Acid 6 and (2) a new condition caused by recessive mutations in the new A/M gene, ALDH1A3 (aldehyde dehydrogenase 1 family, member A3), a key enzyme in the establishment of a retinoic acid gradient along the dorso-‐ventral axis during the early eye development. Materials & Methods: We describe findings in 2 families we identified with STRA6 mutations following molecular analysis of 28 individuals, selected from a cohort of 400+ individuals with A/M according to consanguinity, or relevant features of PDAC. We also review other STRA6 cases. We present the clinical findings in 2 families with mutations in ALDH1A3. Results STRA6: Case 1 had bilateral anophthalmia, tetralogy of Fallot, total anomalous pulmonary venous drainage, small kidneys. The paternal grandfather (heterozygous carrier) had congenital cataract. Case 2, 17-‐week-‐old male fetus, had bilateral diaphragmatic eventration, lung agenesis, hypoplastic left atrium, bilateral anophthalmia and polysplenia. This coupleâ?Ts first pregnancy resulted in a neonatal death of a daughter with bilateral anophthalmia, cryptophthalmos, left pre-‐axial polydactyly bilateral lung agenesis, and a small heart with a ventricular septal defect. ALDH1A3: Case 1 had bilateral extreme microphthalmia, small optic nerves and optic chiasm and autism diagnosed age 3 y. Case 2 had bilateral severe anophthalmia with normal intelligence. Conclusions: Most STRA6 mutations have been identified in individuals with severe A/M together with features of PDAC and a poor prognosis for life expectancy and cognitive development. However, isolated A/M as well as cases with A/M and other non-‐PDAC features e.g. neural tube defect; polysplenia, polydactyly and longer life expectancy are reported. Heterozygous mutations may be associated with milder ocular signs e.g.

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cataract and coloboma Mutations in ALDH1A3 are, so far, associated with bilateral severe microphthalmia, possibly with heart anomalies and autism. The full phenotypic spectrum will emerge when more cases have been identified. 18:30 -‐ MSX2 GENE DUPLICATION WITH EYE DEVELOPMENT DEFECT. JULIE PLAISANCIÉ; ELISE SCHAEFER; CORINNE COLLET; VALÉRIE PELLETIER; HÉLÈNE DOLLFUS, Hôpital Civil, Strasbourg, France Introduction: This case is the first report of ocular abnormalities and limb defects in a patient with a rare syndromic craniosynostosis linked to mutations in the MSX2 gene. The propositus was the first child of healthy and unrelated parents. Her psychomotor development was in the normal range. She presented with a brachycephaly due to bicoronal synostosis. Limb abnormalities were consisting of a left complete and right partial cutaneous syndactyly of the third and the fourth fingers. She exhibited also a left moderate microphthalmia associated with an iridochorioretinal coloboma. No visual impairment was noted for the right eye. Materials & Methods: The molecular analyses realized in this patient disclosed an intragenic duplication of the two first exons of MSX2, after testing all other major genes involved in craniosynostosis. In addition, karyotype analysis of the peripheral blood was 46,XX and the Array CGH did not reveal any anomaly. Results: MSX2 gene is a very rare cause of syndromic craniosynostosis. In the Boston-‐type craniosynostosis, short first metatarsals are classically present and ametropia may occur. Mutations with a gain of function effect on the MSX2 protein have been associated with this phenotype, whereas loss of function mutations in the same gene cause parietal foramina and cleidocranial dysplasia. Conclusions: To our knowledge, this case is the first report of an eye development defect due to a MSX2 gain of function mutation in human. The implication of this gene in eye development has already been shown in animal models. Indeed, overexpression of the Msx2 gene resulted also in optic nerve aplasia and microphthalmia in transgenic mice. This report expands the phenotypic spectrum of the MSX2 mutations and adds to early ocular development knowledge. 18:45 -‐ 20:45 Belgian Beer Tasting

Het Pand Please note that no food will be served -‐ See list of restaurants

Friday August 23, 2013

08:30 -‐ 09:54 Genetics Session 3

Genetic Testing Chairs: Frauke COPPIETERS & Lisa KEARNS

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8:30 -‐ SEARCH FOR NEW GENES IN AUTOSOMAL RECESSIVE RETINITIS PIGMENTOSA. NOUR-‐AL-‐DAIN MARZOUKA; BÉATRICE BOCQUET; MAXIME HEBRARD; ISABELLE-‐ANNE MEUNIER, CHRISTIAN HAMEL, Hôpital Saint Eloi -‐ Bâtiment INM, Montpellier, France Introduction: Autosomal recessive Retinitis Pigmentosa (arRP) is a heterogeneous disorder. To date, 36 genes have been identified to cause arRP, with USH2A and EYS as the causative genes in 10-‐20% of the cases. It is estimated that these 36 genes account for about 60% of the cases, with 40% of the cases thus remaining genetically unidentified. To find new causative genes/loci, we applied a straightforward approach based on using single nucleotide polymorphism (SNP) genotyping followed by sequencing. Materials & Methods: Microsatellite markers specific for the USH2A and EYS genes were used to screen for the homozygosity of these genes in consanguineous families. The negative families from this screening were assayed using 250K SNP microarrays and analyzed by TASE software (Transmitted Allele Search Engine) to identify the homozygous regions on the chromosomes. The exons in the known RP genes belonging to the homozygous regions were amplified and Sanger-‐sequenced. Results: A total of 44 consanguineous families were analyzed. Among them, 14 (32%) were fully or partly homozygous for the EYS or USH2A markers. Sixteen of the 30 remaining families underwent SNP genotyping, homozygosity mapping and subsequent sequencing. We found the causative mutations in known RP genes (RP1, IMPG2, NR2E3, PDE6A, PDE6B, RLBP1, CNGB1, and C2ORF71) in ten (63%) families. Six families do not have mutations of known genes in homozygous regions, which represent possible new loci. Conclusions: Assuming that 32 % of the families have mutations in EYS or USH2A, there is a total of 74 % of the families with mutations in the known RP genes, which is higher than the currently estimated prevalence (60 %). The analysis of candidate loci in the 6 negative families is in progress using WES to identify new arRP genes. 8:42 -‐ GENETICS OF ISOLATED UNILATERAL RETINOBLASTOMA. DIETMANN LOHMAN, Universitatsklinikum Essen, Essen, Germany From 1992 to 2012 we have performed genetic testing in more than 750 patients (pts) with isolated unilateral retinoblastoma. We have analyzed the results to i.) determine the spectrum of molecular etiologies, ii.) define genotype-‐phenotype correlations, iii.) identify the issues relevant for genetic counseling and testing also of family members and to provide the data based on which iv.) optimum strategies for genetic testing can be developed. i.) For more than 500 pts, DNA from fresh frozen tumor and blood DNA was available. In 326 of these pts, DNA of blood showed none of the two RB1 gene alterations present in the tumor (homozygous normal). A mutational mosaic was detected in 32 pts and further 54 pts were found to be heterozygous for an oncogenic mutation. In additional 57 pts an RB1 mutation was detected in blood without prior analysis of tumor DNA. As the methods used for mutation detection evolved over time we ran out of tumor DNA in 38 pts before finishing all analyses as they became available. The Toronto lab showed that a few of our tumors with no RB1 mutations have MYCN-‐amplification instead.

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II.) With respect to somatic mutations there are two genotype-‐phenotype correlations: a) tumors with MYCN-‐amplification were associated with an early age at diagnosis (as reported previously), b) Early age at diagnosis is also observed in some pts with tumors showing retention of the paternal RB1 allele and the RB1 imprint. c) There was no obvious phenotypic feature that correlated with mosaic somatic mutations. Diverse genotype-‐phenotype correlations are observed in pts heterozygous for an RB1 gene mutation: d) almost invariably, pts with gross deletions showed extraocular problems; e) while typical incomplete-‐penetrance type (“mild”) mutations where less frequent than expected some “very mild” changes were observed. iii.) Genetic testing excluded an increased risk for more than 350 families (pts with somatic RB1 and MYCN alterations) with considerable benefits (less eye exams and psychological relief). Incomplete penetrance among parents was less frequent than expected. In some rare families, however, we detected numerous carriers with an increased risk. We have actively approached pts tested in childhood age and find that there a great need for genetic counseling as many of them are misinformed about risk and risk prediction. iv.) With technological advances, specifically next generation sequencing, comprehensive mutation detection in tumors will become available. Moreover, as fewer pts are treated by enucleation there is a need for complete mutation detection, including mosaic cases. From the genotype-‐phenotype correlations it appears that deletion testing in DNA from blood might be an efficient first step of analysis in all children who have extraocular anomalies, specifically muscular hypotonia. 8:54 -‐ A NOVEL APPROACH TO MOLECULAR DIAGNOSIS OF RETINOBLASTOMA USING NEXT GENERATION SEQUENCING AND RB1 CUSTOM ACGH. GEMMA D’ELIA; SIMONA GROTTA; CECILIA SURACE; RAFFAELE COZZA; ADRIANO ANGIONI, Bambino Gesù" Children's Hospital, Rome, Italy Introduction: Retinoblastoma (Rb) is the most frequent intraocular malignant tumor of childhood and results from inactivation of both alleles of the RB1 gene located in 13q14.2. Identification of RB1 mutations is important to complete the diagnostic process and to assess the reproductive risk of the parents. Actually, molecular genetic tests for the study of RB1 include sequencing analysis and copy number variations detection, usually using Multiplex Ligation-‐dependent Probe Amplification (MLPA). Despite the use of these approaches, a significant amount of retinoblastomas escape molecular diagnosis. Moreover, these techniques show some limitations: sequencing is expensive, time consuming and unable to detect low grade mosaicisms; MLPA may generate false positive results and need to be confirmed using other methods. Here, we report our data on molecular diagnosis of Rb using a Next Generation Sequencing (NGS) and RB1 custom Comparative Genomic Hybridization (CGH) array approach. Materials & Methods: A total of 54 patients with retinoblastoma were studied: 29 unilateral, 23 bilateral and 2 trilateral. All samples were analyzed using the NGS platform MiSeq (Illumina) and mutations were reevaluated with conventional Sanger sequencing. Large deletions/duplications as well as small intragenic rearrangements

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were identified by RB1 custom-‐made high-‐resolution oligonucleotide CGH Microarray. Results were confirmed using Real Time PCR or MLPA. Results: Twenty-‐one point mutations were identified in 19 bilateral, one unilateral and one trilateral retinoblastomas. Among genomic abnormalities, five intragenic deletions and four large deletions involving genes adjacent to RB1 were revealed. Moreover, an intragenic duplication from exon 18 to exon 23 was identified. Conclusions: Custom aCGH is able to accurately detect genomic rearrangements of RB1, also allowing to characterize their extension. NGS is easy to perform, cheap and efficient and it is able to find low-‐grade mosaicisms. 9:06 -‐ THE ROLE OF GENETIC TESTING IN ELUCIDATING THE CAUSE OF EARLY ONSET HORIZONTAL NYSTAGMUS (EOHN). JOANNE SUTHERLAND; MEGAN DAY; ELISE HEON, Sick Kids Hospital, Toronto, Canada Introduction: The purpose of this retrospective study is to determine if genetic testing can reduce the amount of time to diagnose the underlying cause of nystagmus that manifests at around 3 months of age. Parents want to know whether to expect a stable or progressive condition and whether other medical issues (syndrome) may develop. Parents can wait months or years before a satisfactory diagnosis is reached. Clarification of the etiology of EOHN typically involves numerous ocular investigations including visual electrophysiology testing. Traditional observations and testing may need to be spaced a year or more apart to determine progression. Identifying mutations in causative genes may shorten the diagnostic odyssey for a significant number of families. Materials & Methods: A FileMaker database was used to review over 600 probands referred to our Pediatric Ophthalmology Clinic for EOHN, close to 30% had molecular testing. Molecular testing was successful for 122 / 183 (67%) probands. Results: Of the 122 EOHN probands with a gene diagnosis: 17 had achromatopsia, 39 had oculocutaneous albinism, 5 had ocular albinism, 10 had congenital stationary nightblindness and 19 had Leber congenital amaurosis for a total of 90 EOHN patients. A diagram of the typical diagnostic timeline will be presented that integrates electrophysiology testing and genetic testing for EOHN. Conclusions: Our success at diagnosing 2/3 of EOHN patients by molecular testing suggests that DNA analysis should be an early part of the new patient workup. Identification of causative mutation(s) within a gene can help direct investigations to come to a diagnostic conclusion in a shorter period of time. A list of genes is identified that play a significant causative role in EOHN. As more genes are able to be tested simultaneously with Next Generation Sequencing, Whole Exome Sequencing (WES) panels and Whole Genome Sequencing (WGS), the cost of genetic testing is decreasing. A molecular strategy for EOHN patients can offer potential savings in health care dollars by reducing the number of ophthalmological tests and examinations as well as providing more specific information to parents in a timely manner. 9:18 -‐ LOSING THE “MIDDLE MAN” IN GENETIC EYE DISEASE: THE CURRENT SCOPE OF DIRECT TO CONSUMER TESTING IN OPHTHALMOLOGY. LISA S KEARNS; MARIA

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FRANCHINA; SEYHAN YAZAR; DAVID A MACKEY; ALEX W HEWITT, University of Melbourne, Royal Victorian Eye & Ear Hospital, East Melbourne, Australia Introduction: Providing personalised genetic information may be seen as “empowering” with individuals taking responsibility for their own health, decisions and lifestyle choices. Although there are regulations covering genetic testing, consumers can approach Direct to Consumer (DTC) companies who provide a variety of tests ranging from paternity and ancestry screening to genetic disease risk assessment. Such services circumvent the direct pre-‐test consultation; thereby removing the “middle man” (medical practitioner or genetic counsellor). This study aims to compare DTC companies and their services who offer genetic risk assessment for ophthalmic diseases. Materials & Methods: Using the Google search engine, DTC company web sites were scanned using the following terms: genetic test; genetic testing; genomic test; DNA test kit; direct-‐to-‐consumer genetic tests; direct-‐to-‐consumer genomic tests; and DTC Company. Those DTC not offering testing for at least one ophthalmological condition in humans were excluded. Information obtained included whether the service was “truly DTC”, cost to the consumer, sample source, eye diseases as well as genes or variants tested. Data were collated on results, interpretation, significance and genetic counselling services provided to the consumer and whether these were updated as additional information became available. Results: Seventeen companies were identified; however, two companies were not used for direct comparison after recent company takeovers. Of the companies compared 10/15 (66.66%) were “true” DTC companies, offering tests to a consumer without the need for a medical practitioner’s input. All companies provided some level of genetic risk assessment for age-‐related macular degeneration. There was variation in SNPs used for a given disease, with varying numbers of SNPs and loci tested. Conclusions: Currently, there is marked variation in genetic screening provided by DTC companies for ophthalmic disease. Such variation in loci and SNPs screened, could lead many patients to receive somewhat different risk predictions if using multiple services. Some level of consumer protection could be ensured through regulation of current DTC services. 9:30 -‐ NEXT GENERATION SEQUENCING PANEL TO DETERMINE THE REAL PREVALENCE FOR GENE DEFECTS UNDERLYING ROD-‐CONE DYSTROPHIES. SAID EL SHAMIEH; KINGA BUJAKOWSKA; JOSE-‐ALAIN SAHEL; ISABELLE AUDO; CHRISTINA ZEITZ, INSERM, Paris, France Introduction: We have developed an unbiased and time-‐efficient retinal gene array using next generation sequencing (NGS). Subsequently, we have enhanced it by improving the coverage of targeted genomic regions and restricting it to the most relevant genes (121 genes). Applying our NGS panel, we have started to analyze 96 of 500 rod-‐cone dystrophy (RCD) patients of whom 89 show arRCD and 7 adRCD. Materials & Methods: We applied a filtering approach against a set of polymorphisms available in public databases. Referenced variants, which do occur with a minor allele frequency â?¥0.005 and â?¥0.001 for gene defects underlying arRCD and adRCD respectively, were removed. We have also stratified candidate mutations based on their

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functional consequences giving a priority to frameshift, nonsense and disruption of canonical splice sites variants. Sanger sequencing and co-‐segregation analyses of available family members were subsequently performed. Results: The overall sequencing coverage of the captured regions was 99% for 25X depth of coverage leaving only a small fraction (0.5%) of uncovered exons. Systematic Sanger sequencing confirmed 97.5% of the mutations identified by NGS. While 57% of arRCD patients were having at least 2 pathogenic variants, 23% were showing one pathogenic variant. Among the unsolved cases showing a single disease-‐causing mutation, 9.5% and 13% were heterozygous for USH2A and CEP290 genes respectively. Regarding the adRCD patients, we were able to explain the phenotype in 72% of total cases. In two patients, we found novel homozygous deletions in RP1. Co-‐segregation analyses pointed out that mutations in RP1 underling autosomal recessive cases are not rare events. Conclusions: Our novel screening method was able to reach significant diagnostic rates, encouraging a future application in clinical diagnosis. Once completed, the current investigation will comprehensively determine the prevalence of mutations in known genes underlying RCD and provide cases with no know gene defect suitable for whole exome sequencing. 9:42 -‐ IDENTIFICATION OF NOVEL HOMOZYGOUS DELETIONS IN CONSANGUINEOUS PEDIGREES AS A SHORTCUT TO CANDIDATE GENE DISCOVERY IN RETINAL DYSTROPHIES. KRISTOF VAN SCHIL; FRANCOISE MEIRE; NICOLAS DECONINCK; THOMY DE RAVEL; SANDRA JANSSENS; ELFRIDE DE BAERE, Ghent University Hospital, Ghent, Belgium. Introduction: Purpose of this study is to identify the underlying genetic cause in 25 pre-‐screened consanguineous families diagnosed with autosomal recessive retinitis pigmentosa (ARRP) or Leber congenital amaurosis (LCA) using identity-‐by-‐descent (IBD) mapping and to demonstrate the power of mapping of homozygous deletions as a shortcut to gene identification in retinal dystrophies (RDs). Materials & Methods: IBD mapping was performed by genome-‐wide SNP chip analysis (HumanCytoSNP-‐12, Illumina). For IBD data analysis we integrated PLINK with arrayCGHbase, a platform for data analysis of microarray based comparative genome hybridization. Deletions were confirmed and fine-‐mapped by conventional PCR. Segregation analysis was performed by qPCR (LightCycler, Roche; qBaseplus, Biogazelle). Results: Homozygous deletions were identified in 3 out of 25 families. The first deletion (133 kb) was found in an ARRP patient and removes the first non-‐coding exon of the known gene EYS. The second deletion (1,3 Mb) was found in a patient with Usher syndrome type I. It disrupts the first non-‐coding exon of PCDH15 and the single-‐exon gene MTRNR2L5. The third one (331 kb) is a partial deletion of GRID2 (E2), found in a patient with LCA and vermis atrophy. It leads to an in-‐frame deletion (p.Gly30_Glu81del). All of them were located in the one but largest IBD region. The deletions could be confirmed and segregation could be demonstrated. There have been reported disease-‐causing CNVs in the coding region of EYS in patients with ARRP (Pieras et al. IOVS 2011), non-‐coding deletions of EYS however have not yet

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been described. As to PCDH15, a similar non-‐coding deletion has already been described, although not involving the MTRNR2L5 gene (Le GuÃ©dard et al., 2007). GRID2 encodes a neurotransmitter receptor that plays an important role in the brain and is also regulated by the retinal transcription factor CRX. Hotfoot mice with similar homozygous deletions in this gene are viable and develop cerebellar ataxia (Lalouette et al., 1998). Recently, a homozygous deletion in GRID2 (E3-‐4) has been reported for the first time in human (Utine et al., 2013), displaying a similar neurological phenotype. Conclusions: This study revealed involvement of a homozygous promoter and 5â?TUTR deletion of EYS in ARRP and PCDH15 in Usher syndrome, and uncovered a potential novel candidate gene for RD, GRID2. We demonstrated that homozygous deletion detection in consanguineous families might be a powerful approach for elucidation of novel mechanisms and genes for RDs. 09:55 -‐ 10:40 François Lecture: "Fibrillinopathies and

related disorders -‐ Molecular considerations and ocular phenotypes"

Dr. Elias I TRABOULSI, Cleveland, OH, USA Introduced by Dr. Bart P. LEROY


11:15 -‐ 13:00 Symposium: Update on Gene Therapy & Stem Cell Therapy for Retinal Dystrophies

Chairs: Mark PENNESI & Bart P LEROY

11:15 – HOW DO YOU DELIVER GENE THERAPY TO THE EYE. ALBERT M. MAGUIRE, Philadephia, PA, USA 11:30 -‐ RETINAL DYSTROPHIES: DEFINING NEW ENDPOINTS FOR THERAPEUTIC TRIALS. BIRGIT LORENZ, Department of Ophthalmology, Justus-‐Liebig-‐University Giessen, Germany Gene therapy has the potential to become a realistic therapeutic option for the treatment of inherited retinal dystrophies. However, the importance of obtaining objective and measurable data to show a real clinical benefit is evident. With the availability of highly sensitive morphological and functional examination techniques, new clinical endpoints should be defined that clearly differentiate between clinical benefit and placebo effect. Morphological parameters include data on intraretinal layer thickness and alterations in the appearance of specific layers, as well as fundusautofluorescence data. Functional parameters include the definition of the light sensitivity threshold using the full field stimulus test or the 2-‐color threshold perimetry, as well as chromatic pupillometry or fundus controlled perimetry and dark adaptation.

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This presentation will present some of the recent data obtained with these techniques on patient cohorts that could be enrolled in current gene therapy trials. 11: 45 – GENE THERAPY FOR LCA. JEAN BENNETT. Philadelphia, PA, USA 12:00 – GENE THERAPY FOR CHOROIDEREMIA. ALUN BARNARD, Oxford, UK 12:15 – GENE THERAPY FOR STARGARDT DISEASE. ISABELLE AUDO, Paris, France 12:30 – GENE THERAPY FOR UNSHER SYNDROME TYPE 1B. MARK PENNESI, Portland, OR, USA 12:45 – STEM CELL THERAPY FOR RETINAL DYSTROPHIES. MANDEEP SINGH, Oxford, UK 13:00 – 14:00 Lunch -‐ Provided on Site

Poster Viewing

14:00 -‐ 14:30 Business Meeting of ISGEDR All Members David MACKEY Presiding

14:30 -‐ 15:00 Genetics Session 4 Therapy for retinal dystrophies

Chairs: Arlene DRACK & John G FLANNERY

14:30 -‐ AAV-‐RDCVF RESCUES CONES AND AAV-‐RDCVFL PROTECTS RODS IN RETINAL DEGENERATION. JOHN G. FLANNERY; LEAH C. BYRNE; DENIZ DALKARA; GABRIEL LUNA; STEVEN K. FISHER, University of California, Berkeley, USA Introduction: The bifunctional gene Nxnl1 encodes two isoforms of the rod-‐derived cone viability factor (RdCVF) through alternative splicing. RdCVF, a truncated thioredoxin-‐like protein, is secreted from rods and has been shown to have a protective effect on the survival of cones, while the full length isoform, RdCVFL, contains a thioredoxin fold and may be involved in oxidative signaling and protection against hyperoxia. We evaluated the protective effects of AAV-‐mediated expression of these two isoforms in the rd10 mouse model of rod-‐cone dystrophy Materials & Methods: Expression of RdCVF and RdCVFL was accomplished via intravitreal and intravenous injection of engineered AAV vectors. 7m8, a novel AAV variant, was injected intravitreally, and AAV92YF was delivered systemically to provide earlier onset of expression than possible by intraocular injection. Results: Intravitreal AAV-‐RdCVF slowed the rate of cone cell death and decreases in the photopic electroretinogram compared to the contralateral control eye. Early expression of RdCVFL but not RdCVF increased levels of rhodopsin mRNA and reduced biproducts of oxidative stress. Expression of AAV-‐RdCVF earlier in the degeneration via systemic

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administration led to longer cone survival and concomitant preservation of cone physiological function. Conclusions: Expression of the two isoforms of RdCVF has positive, disparate effects on rods and cone survival. Expression of RdCVF leads to structural and functional rescue of cone photoreceptors, but has little effect on rods whereas RdCVFL expression alone did not significantly rescue cones. Coexpression of RdCVF and RdCVFL heightened the observed rescue effect on all photoreceptors. In contrast, expression of RdCVFL early in the course of disease in dark reared rd10 animals prolongs rod function, increased levels of rhodopsin and decreased the byproducts of cellular oxidative stress. These results indicate that RdCVF and RdCVFL protect photoreceptors through separate complementary mechanisms and show proof-‐of-‐concept for a widely applicable viral vector-‐mediated gene therapy that has the potential to prolong vision in patients. These experiments demonstrate the efficacy of AAV vectors as a delivery strategy for RdCVF, and suggest that expression of RdCVF is a promising approach to delaying the loss of cones in patients with rod-‐ cone dystrophy. Viral vectors could potentially be used to express the protein for the life of the patient following a single treatment. AAV-‐mediated delivery of RdCVF could potentially benefit patients carrying any mutation underlying rod-‐cone dystrophy, making this therapy a flexible approach to prolonging vision in a spectrum of disorders. 14:45 -‐ IMMUNOSUPPRESSIVE THERAPY FOR RETINAL DEGENERATION IN BATTEN DISEASE. ARLENE DRACK; ROBERT MULLINS; ERIKA AUGUSTINE; WANDA PFEIFER; SANDY HONG, University of Iowa, Iowa City, IA, USA Introduction: Batten disease, or Juvenile Neuronal Ceroid Lipofuscinosis, caused by mutations in the CLN3 gene, is a fatal neurodegenerative condition of childhood. Formerly healthy, developmentally normal children present with progressive vision loss at 5 to 7 years of age. Blindness usually results within 2 years, followed by inexorable neurodegeneration.There is no treatment or cure. The immune response has been postulated to play a role in the neurodegeneration. Materials & Methods: Case report of a child with genetically confirmed Batten disease1 treated with systemic (mycophenolate mofetil 600mg/m2) and local (subtenon and topical steroid, topical Nefanac) immunosuppression for 2 years. Complete eye examinations including color vision testing, optical coherence tomography, electroretinography (ERG), Goldmann Visual Field (GVF) and vitreous cell photography were performed every 3-‐4 months. Results: Central visual acuity was 20/400 right eye and 20/300 left eye at the start of treatment and 20/500 right eye and 20/500 left eye 2 years later. Dark-‐adapted standard combined response ERG amplitudes were stable, however 30 Hz flicker responses went from barely recordable to non-‐recordable. GVF demonstrates retention of the V4e isopter. Anterior vitritis lessened over time, more in the eye first treated locally. A small pericentral posterior subcapsular cataract developed in that eye. Mycophenolate mofetil has been discontinued several times due to acute illness with fever, and later restarted without adverse events.

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Conclusions: In the mouse model of Batten disease, as well as in humans with the disease, anti-‐GAD antibodies have been detected2. In the mouse model, modulating this neuro-‐inflammatory response resulted in better performance on the rotarod test3, and a short term clinical trial in humans is currently underway based on this data (www.clinicaltrials.gov). Due to an intense vitreous inflammatory response, our patient has been treated aggressively with systemic and local immunosuppression for 2 years, and still has ambulatory vision and a recordable dark adapted standard combined response ERG 3 years after vision loss was first documented, which is unusual for Batten disease retinopathy4. Immunosuppression may favorably alter the course of retinal degeneration in Batten disease. References: 1. Drack, Miller, et al. J Clin Neurol in press 2. Pearce et al. Neurol 2004. 3. Sheehan et al. J Neuroimmunol. 2011 4. Bozorg et al. Surv Ophthalmol 2009. 15:00 -‐ 16:00 Symposium

Genome Wide Association Studies (GWAS) Chairs: Christopher HAMMOND & David MACKEY

15:00 -‐ OPHTHALMIC SUCCESS WITH GENOME WIDE ASSOCIATION STUDIES (GWAS). DAVID A MACKEY, Lions Eye Institute, University Of Western Australia GWAS have proven to be highly effective in identifying new disease genes. In ophthalmology CFH, LOXL1 and TCF4 are outstanding examples of opening understanding for Age-‐Related Macular Degeneration, Exfoliation Syndrome and Fuch's Corneal Dystrophy. Large meta analyses relating to glaucoma and myopia are similarly productive. 15:15 – AN INTERNATIONAL GWAS OF GLAUCOMA-‐RELATED OPTIC DISC PARAMETERS: THE INTERNATIONAL GLAUCOMA GENETICS CONSORTIUM. HENRIËT SPRINGELKAMP. Rotterdam, The Netherlands 15:30 – GWAS OF REFRACTIVE ERRORS. CHRISTOPHER HAMMOND. LONDON, UK 15:45 -‐ Genome-‐wide association studies of central corneal thickness and keratoconus. SEYHAN YAZAR, Centre for Ophthalmology and Visual Science, University of Western Australia, Lions Eye Institute Central corneal thickness (CCT) is a quantitative trait that is associated with eye conditions including keratoconus and glaucoma. In a large meta-‐analysis including more than 20,000 individuals of European and Asian ancestry, we identified 16 novel CCT-‐associated loci at genome-‐wide significance level (P < 5 × 10−8). Two CCT-‐associated loci, FOXO1 and FNDC3B, were conferred relatively large risks for keratoconus in 874

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cases and 6,085 controls. Additionally, FNDC3B was found to be associated with primary open-‐angle glaucoma. These genes are involved in collagen and extracellular matrix pathways that may explain their association with CCT. 16.00 -‐ 16.30 Break

Poster Viewing

16.30 -‐ 17:54 Genetics Session 5 Multifactorial Genetics & Behavioural Studies


16:30 -‐ ROLE OF GENETIC LOCI IMPLICATED IN DIABETIC RETINOPATHY RISK. ANNIE MCAULEY; JIE JIN WANG; PAUL CONNELL; MOHAMED DIRANI; ALEX HEWITT, Centre for Eye Research Australia, University of Melbourne, Royal Victorian Eye & Ear Hospital, Victoria, Australia Introduction: Recent genome-‐wide association studies (GWAS) for diabetic retinopathy (DR) have identified nineteen novel single nucleotide polymorphisms (SNPs) associated with DR in type 1 diabetes in Caucasian populations, and in a separate study eight different loci were nominally associated with DR in type 2 diabetics in an Asian population. These suggestive associations have not been well validated in independent cohorts. The objective of this study was to ascertain any association of these SNPs with sight threatening DR in an Australian cohort. Materials & Methods: A total of 592 Australian participants who had either type 1 (n=109) or 2 (n=483) diabetes were genotyped for 27 SNPs previously identified in association with DR. Cases (n=163) were defined as people with severe non-‐proliferative DR (NPDR) or proliferative DR (PDR). Control participants (n=368) had either no evidence of DR, or only mild NPDR as defined by the Early Treatment of Diabetic Retinopathy Study protocol using MESA-‐EYE Digital Grading guidelines. Evaluation of associations between case and controls were conducted using logistic regression analysis for allele frequencies, with co-‐variable adjustment. Sub-‐analysis with 483 type 2 diabetic subjects was also undertaken. Bonferroni correction was used for significance testing (p=0.05/27=0.0019) in all analyses. Results: In a combined analysis of type 1 and 2 DM we observed a nominal associated between PDR/severe NPDR with alleles rs1073203-‐G (p=0.009, OR=0.31, 95% CI, 0.13-‐0.74) and rs476141-‐A (p=0.05, OR=0.62, 95% CI, 0.38-‐1.00); however, the associations were not significant following Bonferroni correction. Sub-‐group analysis in type 2 DM also revealed no significant associations with severe NPDR or PDR. Conclusions: This study did not replicate the findings of recently identified SNPs associated with sight-‐threatening DR within our Australian cohort. Therefore, these SNP candidates may not be appropriate for DR risk assessment within an Australian population, and future strategies should be population specific.

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16:42 -‐ NO EVIDENCE FOR DIFFERENTIAL IL17RC PROMOTER METHYLATION IN AGE-‐RELATED MACULAR DEGENERATION. MARIA FRANCHINA; BRENDAN J. VOTE; JAMIE E. CRAIG; DAVID A. MACKEY; ALEX W. HEWITT, Lions Eye Institute, Western Australia, Australia Introduction: Age-‐related macular degeneration is a leading cause of visual impairment worldwide. The pathogenesis of AMD involves unknown complex interactions between genes and the environment. DNA methylation is an important epigenetic mechanism by which gene expression can be altered, such as in response to an environmental stimulus. Aberrant DNA methylation within the promoter of IL17RC, a gene which encodes a single-‐pass type I transmembrane protein involved in T-‐cell activation, has recently been identified in AMD. To further investigate the epigenetic dysregulation in AMD, we mapped the methylation status of the IL17RC promoter within an Australian cohort of AMD patients and age and gender matched controls. Materials & Methods: All patients and controls were genotyped for the CFH His402Tyr (rs1061170) variant on chromosome 1q31 and the ARMS2 Ala69Ser (rs10490924) variant on chromosome 10q26 which are strongly associated with AMD using iPLEX Gold chemistry (Sequenom Inc., San Diego, California) on an Autoflex mass spectrometer at the Australian Genome Research Facility, Brisbane, Australia. Cytosine methylation status at 8 CpG dinucleotides within the IL17RC promoter was determined by bisulphite pyrosequencing using the PyroMark Q24 Sequencer (Qiagen, Maryland, USA). The percentage methylation at each CpG dinucleotide between AMD patients and controls was compared using Wilcoxon signed ranks test. Results: We initially confirmed the previously reported associations between the CFH His402Tyr polymorphism (rs1061170) and the ARMS2 Ala69Ser polymorphism (rs10490924) and presence of AMD in our cohort. On bisulphite genomic pyrosequencing, we did not detect any significance differences in methylation status at any of the CpG dinucleotides analysed within the IL17RC promoter in AMD patients compared to the controls. Conclusions: Our findings indicate that hypomethylation within the IL17RC gene promoter is not pathognomonic of age-‐related macular degeneration. 16:54 -‐ ASSOCIATION OF GENETIC RISK FOR REFRACTIVE ERROR AND TIME SPENT OUTDOORS. SEYHAN YAZAR; GABRIEL CUELLAR; CHARLOTTE M. MCKNIGHT; ALEX W. HEWITT; STUART MACGREGOR, Centre for Ophthalmology and Visual Science, University of Western Australia, Western Australia, Australia Introduction: Myopia has reached epidemic levels in East Asian populations over the past two decades leading to increased efforts to identify the genetic and environmental factors that confer risks for development of this refractive error. Recently, multiple genes were found to be associated with myopia in two separate large genome-‐wide association studies. In parallel to this genetic progress, many epidemiological studies have shown that that reduced time outdoors is a risk factor for myopia. In order to understand the gene-‐environment interaction in development of myopia we aimed to investigate the association between time spent outdoors and genetic predisposition to myopia.

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Materials & Methods: At the 20-‐year follow-‐up 1344 participants of the Western Australian Birth Cohort underwent a comprehensive eye examination including cycloplegic autorefraction. We also determined the amount of conjunctival ultraviolet autofluorescence (UVAF) as a measure of time spent outdoors. Genotype data and consents for genetic studies were available from the previous assessments. We calculated a genetic risk score based on susceptibility SNPs for refractive error previously discovered by the Consortium for Refractive Error and Myopia (CREAM). Refractive error was measured by spherical equivalence (SE). Myopia was defined as mean SE of both eyes equal to or less than -‐0.50 diopters (D). Results: In a multivariate model adjusted for age and gender, the genetic risk score (p<0.001) and UVAF (p=0.033) were independently associated with refractive error. A more progressive increase in refractive error with increasing genetic risk score was observed among participants with low levels of UVAF (Î²=1.03) compared to those with high levels of UVAF (Î²=0.54). The odds of being non-‐myopic with high genetic risk score and high levels of UVAF was 6.09 (95%CI: 1.92 -‐ 27.14) times higher than the odds of being non-‐myopic with low genetic risk score and low levels of UVAF (p=0.006). Conclusions: Our findings show that the genetic predisposition to refractive error is influenced by time spent outdoors. High genetic susceptibility with less time spent outdoors may trigger development of myopia. 17:06 -‐ ENRICHMENT OF PATHOGENIC ALLELES IN THE BRITTLE CORNEA GENE, ZNF469, PROVIDES NOVEL MOLECULAR INSIGHTS INTO THE DEVELOPMENT OF CORNEAL THINNING AND KERATOCONUS. LOUISE PORTER; JUDITH LECHNER; AINE RICE; GRAEME BLACK; COLIN WILLOUGHBY, University of Manchester and Manchester Royal Eye Hospital, UK Introduction: Keratoconus, a common inherited ocular disorder resulting in progressive corneal thinning is the leading indication for corneal transplantation in the developed world. Genome-‐wide association studies have identified common SNPs near ZNF469 strongly associated with corneal thickness. Homozygous mutations in ZNF469 and PRDM5 genes result in brittle cornea syndrome. Brittle cornea syndrome is an autosomal recessive connective tissue disorder associated with extreme corneal thinning and a high risk of corneal rupture. Individuals with heterozygous mutations in brittle corneal syndrome genes often demonstrate a carrier ocular phenotype displaying reduced corneal thickness and keratoconus. We hypothesize that heterozygous variants in PRDM5 and ZNF469 predispose to the development of isolated keratoconus. Materials & Methods: The coding region and intron-‐exon junctions of PRDM5 were sequenced by Sanger sequencing in 96 unrelated patients with keratoconus. ZNF469 was sequenced in a cohort of 153 unrelated patients with keratoconus from three study centers (Belfast, Leeds and Lausanne). We detected sequence variants as potentially disease-‐associated alleles by filtering using sequencing data from 96 control subjects as well as data from dbSNP (Build 137), May 2012 release of the 1000 Genomes (1KG) Project and the Exome Variant Server (EVS), NHLBI Exome Sequencing Project (ESP), with no allele having a minor allele frequency of > 0.1%. We considered only non-‐synonymous alleles which were conserved when analyzed using Homologene and

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multiple sequence alignment with ClustalX; and those deemed damaging on either the Polymorphism Phenotyping v2 (PolyPhen-‐2) and Sorting Intolerant from Tolerant (SIFT) algorithms. We also looked at segregation of variants in a panel of familial keratoconus subjects. The collapsing method was used to compare the frequency of rare alleles between case and control subjects with the level of significance set to p < 0.05. The estimated odds ratio (OR), relative risk (RR), 95% confidence interval (CI) and Fisher exact p-‐value were calculated. Results: This analysis failed to identify any pathogenic variants in PRDM5 in keratoconus subjects. In ZNF469 there were 21 pathogenic non-‐synonymous alleles in the keratoconus subjects and 2 in the controls, which represents a significant enrichment (p < 0.0001) resulting in a relative risk of 11.89. Conclusions: This enrichment of rare pathogenic alleles in ZNF469 in 11.8% of keratoconus patients represents a significant mutational load. It highlights ZNF469 as the most significant genetic factor responsible for keratoconus identified to date. 17:18 -‐ DESCRIPTION OF THE INTELLECTUAL, ADAPTIVE, AND BEHAVIOURAL FUNCTIONING OF PATIENTS AFFECTED WITH BARDET-‐BIEDL SYNDROME: A PRELIMINARY REPORT. ELISE HEON; APARNA BHAN; ELISEZABETH KERR, The Hospital for Sick Children, University of Toronto, Ontario, Canada Introduction: Patients with Bardet-‐Biedl syndrome (BBS; MIM209900) suffer from a multisystem debilitating disorder characterized by digit anomalies, retinal degeneration, genital and kidney malformation/function, obesity, and cognitive impairment. These patients are also known to have a variable degree of cognitive impairment and emotional lability. Anxiety, depression and a range of autistic and obsessive compulsive behaviors have been described but poorly characterized. We prospectively assessed molecularly confirmed BBS patients using a standardized battery of neuro-‐psycho cognitive tests. Materials & Methods: We prospectively evaluated 10 patients (mean age=18 years). Patients’ intellectual functioning was characterized using 2 categories of tests; WAIS-‐II and SIB-‐R. Informant report of behavior (BASC and ASRS) was also objectively defined. Results: The amount of information extracted from each assessment varied based of the degree of visual and or cognitive impairment. The mean verbal reasoning abilities fell at the upper end of the borderline range (mean Standard Score=79.1 SD=19.33). Most patients performed better when defining words (mean scaled score=7.6, SD=3.9) than in abstract verbal reasoning (mean scaled score=5.0, SD=2.7), which respectively represent low average and borderline group performance. The mean perceptual reasoning abilities fell in the EL range (mean Standard Score=66, SD=12.94), with 1/10, 2/10 and 5/10 respectively performing in the low average, borderline, and extremely low ranges. Two individuals were unable to complete the measures. Parent ratings of Overall Adaptive Independence was extremely low (mean SS=41.8, SD=16.9). Extremely low group mean performance was also documented on all subdomains; namely, motor, social/communication, personal-‐living, and community-‐living skills. The most variability was documented in social/communication skills. Using a

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behavioral questionnaire related to everyday life, parents’ mean rating of depression, withdrawal, and atypical behavior were elevated. On an autism spectrum rating scale (using normative data for 12 to 18 years of age) the BBS group means for social-‐communication, unusual-‐behaviors, and self-‐regulation scales fell in the slightly elevated, elevated, and average ranges respectively, with their mean group ratings for subdomains related to peer socialization, social/emotional reciprocity, and sensory sensitivity being elevated and those for atypical language, stereotype behaviors, behavior rigidity being slight elevated. Group mean ratings for attention and adult socialization were average. Testing of additional patients is ongoing. Conclusions: Further characterization of the neuro-‐cognitive aspects of this condition contributes to better understanding the phenotype and provides additional outcome measures. Furthermore this has been of great help for families in better understanding their child and the debilities related to the condition. 17:42 -‐ 19.15 Poster session All authors required to

stand by poster

19:15 -‐ bedtime

Free Evening in Ghent See list of restaurants

Saturday August 24, 2013

8:30 -‐ 09.54 Retinoblastoma Session 2 Treatment

Chairs: Ahmad SAMIR & Marcus DE JONG

8:30 -‐ Vitreous seeding after management with chemoreduction and focal therapy for bilateral retinoblastoma. NATALIA MATTI; Mexican Social Security Institute (IMSS), Mexicali Baja California, Mexico (ISGEDR TRAVEL AWARD RECIPIENT) Purpose: To report our experience in the management of Bilateral Retinoblastoma with chemoreduction and focal therapy. Methods: In an effort to preserve eye and vision function, in 2008 the Pediatric Oncology Department of the Hospital General de Tijuana, Mexico began treating patients with Bilateral Retinoblastoma (whom before were treated with bilateral enucleation) with chemoreduction and tumor consolidation using focal laser photocoagulation, cryotherapy or thermotherapy. Two male patients with bilateral retinoblastoma (4 eyes with 7 tumors) received intravenous chemoreduction with 6 cycles of vincristine sulfate, etoposide and carboplatin combined with focal therapy. After the 4th chemotherapy cycle the tumors were surrounded with laser in order to destroy their blood supply. A second laser session was given a month after. New tumors were treated with additional laser, cryotherapy or thermotherapy as needed.

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Results: The first patient was diagnosed at three months of age and presented group A Rb in the right eye and group C Rb in the left eye. Tumors in the right eye presented regression, but enucleation was performed in the left eye because five additional tumors with extensive vitreous seeding were observed during chemoreduction. Eleven months later the right eye presented a new intraretinal tumor near the ora serrata which was treated with cryotherapy and showed regression. Two years later vitreous seeding was observed next to a flat chorioretinal scar near the superior temporal arcade. The second patient was diagnosed at 5 months of age. He presented group A Rb in the right eye and group E Rb in the left eye. After chemoreduction the left eye was enucleated because of total retinal detachment and extensive subretinal and vitreous seeding. Tumors in the right eye responded to treatment showing type 3 and 4 regression. A new intraretinal tumor was observed 18 months later and was treated with thermotherapy. One month later vitreous seeding was observed. Both patients are now being treated with external beam radiotherapy. Significance: These were the first patients with bilateral retinoblastoma treated with chemoreduction and focal therapy at our hospital. A big effort was made by the Pediatric Oncology and Ophthalmology departments in order to try and preserve eyes and vision. Both patients needed enucleation in one eye and presented new intraretinal tumors and vitreous seeding after chemoreduction and focal treatment. Treating these patients has been a challenge but we are committed to continue doing so and not choosing enucleation as the treatment of choice for patients with bilateral retinoblastoma. 8:42 -‐ FOCAL TREATMENT OF RETINOBLASTOMA TUMORS WITH SIMULTANEOUS 810NM AND 532NM LASERS. ASHWIN MALLIPATNA; VANDHANA SUREN; SUPRIA DABIR; Narayana Nethralaya Bangalore, Karnataka, India Introduction: Focal laser therapy is an essential aspect of treatment of intraocular retinoblastoma, with the utilization of multiple types of lasers and methods of delivery. The effect of the 810nm laser and 532nm laser are different, and simultaneous use of both lasers on a single tumor in the same treatment session is not common. This study describes the results of using this combination of lasers on tumor response. We describe our rationale and experience in utilizing the effects of a combination of both lasers in an attempt to achieve a more rapid reduction in tumor size. Materials & Methods. We retrospectively analyzed the simultaneous use of the 810 and 532nm lasers delivered through the same laser indirect ophthalmoscope in 14 children with intraocular retinoblastoma. Results: A total of 32 tumors were individually analyzed in 18 eyes of 14 children with intraocular retinoblastoma. An initial gentle application of 810nm laser rendered the tumor opaque, without affecting the tumor vasculature. Once the tumor vasculature was clearly visible, application of the 532nm laser was able to photocoagulate this tumor vasculature. With this technique, we were able to demonstrate rapid reduction in tumor size in specific instances, along with documentation of the effect of each laser with ocular coherence tomography. None of the cases experienced laser related

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complications after the combination of lasers. Twenty-‐ eight tumors went on to be inactive. Three eyes required enucleation for persistent tumor activity. Conclusions: A combination of 810nm and 532nm lasers used in the same session might help augment the effect of focal laser therapy. We would suggest this technique be used in certain situations to help a rapid reduction in tumor size. Caution must be exercised to ensure that we do not over apply laser, leading to immediate and delayed laser related retinal complications. 8:54 -‐ STANDARD CONSERVATIVE APPROACHES USING SYSTEMIC CT AND LOCAL TREATMENTS EXPERIENCE OF THE INSTITUT CURIE. ISABELLE AERTS, LIVIA LUMBROSO-‐LE ROUIC, ALEXIA SAVIGNONI, LAURENCE DESJARDINS; Department of Oncologic Pediatry, Institut Curie, Paris France Introduction: Until the early 90’s, the only conservative treatment of RB with posterior pole tumors was external beam radiotherapy (EBR). Currently, intraocular retinoblastoma treatments often associate chemotherapy and focal treatments. The protocols vary and may combine two or three drugs, and different number of cycles associated to the local adjuvant treatments. A first prospective protocol of conservative treatments in our institution showed the efficacy of the use of two courses of chemoreduction with etoposide and carboplatin, followed by chemothermotherapy using carboplatin, as a single agent. We recently review all consecutive patients treated between 1995 and 2009 in our institution. Materials and methods: We review all the retinoblastoma cases treated in our institution between 1995 and 2009 and analyze the response to conservative approach. We considered as failure of the conservative approach, the need for secondary enucleation or EBR. Results: Between 1995 and 2009, 728 patients (pts) were treated in Institut Curie: 411 unilateral and 317 bilateral. Familial history was found in 17.7% in bilateral cases and 2.9% in unilateral. For the unilateral RB, median follow-‐up was 61 months [0-‐198] with a median age at diagnosis of 20 months [0-‐131]: 87 pts were treated with a conservative intention. A successful conservative approach without EBR or secondary enucleation was observed in 64/87 patients (70%). No toxic or disease related death, no extraocular relapses were observed. For the 317 bilateral cases, median follow-‐up was 72 months [0-‐210] with a median age at diagnosis of 7 months [0-‐59]. A successful conservative treatment of at least one eye, avoiding EBR and enucleation was achieved in 70% of the patients. Extraocular disease was observed in 3 pts (all with advanced initial disease: buphtalmia and/or IRSS stage III). 8 Cases of second primary tumors are registred to date (pinealoblastoma: 2 pts, sarcoma: 2 pts, other second tumours: 4 pts). Six patients are dead (2 from pinealoblastoma, 2 from extraocular disease, 2 after palliative approach in the context of 13q deletion with severe handicap). Conclusion: Chemotherapy and local ophthalmologic treatments can avoid EBR or enucleation in approximately 70% of the patients without major toxicities. Survival rate after conservative treatment is 99%. Evaluation of the incidence of second primary cancer needs a longer follow –up.

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9:06 -‐ MANAGEMENT AND OUTCOME OF 550 CASES OF RETINOBLASTOMA TREATED AT A TERTIARY REFERRAL CENTER IN INDIA. BHAVNA CHAWLA; RAJVARDHAN AZAD; RACHNA SETH; SEEMA SEN SHALINI GUPTA, All India Institute of Medical Sciences, New Delhi, India Introduction: The purpose of this study is to report the clinical presentation, management and outcome of retinoblastoma in a large case series from a single institution in India Materials & Methods: Five hundred and fifty consecutive patients of retinoblastoma were evaluated for demographic details, clinical features and outcome following treatment. Retinoblastoma staging was done as per International Classification. Depending upon the tumor stage, treatment strategies included focal therapy and chemoreduction for globe salvage and primary enucleation for advanced intraocular disease. Adjuvant chemotherapy was advised if high-‐risk histopathologic features were present.Extraocular retinoblastoma was treated with a combination of neoadjuvant chemotherapy, enucleation and external beam radiotherapy. Results: Of 550 children, 60% were males. The median age at diagnosis was 24 months. Thirty one percent patients had bilateral tumors. The median follow-‐up was 30 months. Twenty eight percent cases had extraocular spread at initial presentation, which was confirmed on MRI of the orbits and brain. Treatment modalities consisted of enucleation in 66%, systemic chemotherapy in 59%, focal therapy in 25% and external beam radiotherapy in 12% cases. High risk histopathologic features were present in 40% of enucleated eyes. The Kaplan-‐Meier event free survival at 2 years follow-‐up was 87%, with a statistically significant difference between intraocular and extra-‐ocular cases (p=0.000). Conclusions: In India, the majority of patients present with advanced intra-‐ocular disease or with overt extra-‐ocular spread, reflecting the need for early referral. The use of multi-‐modal treatment approach has led to better survival rates in retinoblastoma with late presentation. 9:18 -‐ EXTERNAL BEAM RADIATION THERAPY FOR RETINOBLASTOMA THAT WAS RESISTANT TO CHEMOTHERAPY AND FOCAL TREATMENT: OUTCOME AND PREDICTIVE FACTORS. YACOUB A. YOUSEF, King Hussein Cancer Center, Amman, Jordan

CANCELLED Purpose: To evaluate the outcome and the predictive factors of external beam radiation therapy (EBRT) for treatment of retinoblastoma that was resistant to chemotherapy and focal therapy. Methods and Materials: A retrospective clinical case series of 24 eyes in 20 retinoblastoma patients treated by EBRT, after failure of tumor control by chemotherapy and focal therapy. The main outcome measures include: demographic information, laterality, family history, age at diagnosis, initial tumor stage, tumor seeding, modalities of treatment, post radiation complications, eye salvage, visual acuity, metastasis, second malignancy, and survival. Results: The mean age at diagnosis was 20 months (median, 12 months; range, 2–48 months). There were 12 males (60%) and 8 females (40%). There were 4 (20%) unilateral

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and 16 (80%) bilateral cases. All eyes were treated initially by systemic chemotherapy (CVE) with mean 7 cycles (median, 8 cycles; range, 6-‐8 cycles) with focal therapy (transpupillary thermotherapy and/or cryotherapy). The standard dose of 45Gy was used in all treated eyes. Eye salvage after EBRT was achieved in11 eyes (45%) at a mean follow-‐up 55 months (median, 42 months; range, 12-‐140 months); in 67 % (2/3) of group B, 63 % (5/8) of group C, and 31 % (4/13) of group D eyes. All treated unilateral cases were subsequently enucleated. Vitreous seeds at time of EBRT and tumor stage migration during management by chemotherapy were the most important significant predictive factors for tumor control; (p= 0.0327 and 0.0333 consecutively). Patient’s gender, laterality, type and number of cycles of chemotherapy, and initial RE stage were not significant predictors of the outcome. The overall ocular complications rate was 80% (19/24), and visual acuity was less than 20/400 in 45% of salvaged eyes. Conclusions: EBRT was effective in resistant cases to chemotherapy and focal therapy in absence of the poor predictive factors; vitreous seeds at time of radiation therapy and stage migration during chemotherapy. The side effects of EBRT may not be justified when the eye had poor prognostic factors mainly when the other eye has good vision and tumor control. Enucleation rather than EBRT is recommended in unilateral cases after failure of chemotherapy. 9:30 -‐ TRILATERAL RETINOBLASTOMA AT THE AGE OF NINE WEEKS. VICKTORIA (VICKY) VISHNEVSKIA-‐DAI; IRIS MOROZ; YORAM NEUMANN; AMOS TOREN; JOSEPH MOISSEIEV, Sheba Medical Center, Tel Aviv University, Israel Introduction: Midline intracranial primitive neuroectodermal tumor in the pineal or suprasellar region. TRb occurs in 1.5%â?"5% of patients with either unilateral or bilateral hereditary retinoblastoma. Median age at diagnosis of retinoblastoma is 5 months. Suprasellar TRb usually is diagnosed earlier, than pineal TRb. Patients with TRb have a poor prognosis, mainly due to spreading to the sub-‐arachnoid space and leptomeningeal tumor dissemination. The mean length of survival is 9.7 months in patients who undergo treatment and 1.3 months in those who do not. Effective treatment of trilateral retinoblastoma may require aggressive chemotherapy treatment and close evaluation of for leptomeningeal dissemination. Our Purpose is to report of the management of a 9 weeks old baby with Trilateral Retinoblastoma. Materials & Methods: Case report : A 9 weeks old male baby second of twins, a product of normal birth post 36 weeks of gestation, with no family history of retinoblastoma was presented with bilateral retinoblastoma simultaneously associated with pineal gland tumor. At the age of six weeks the mother noticed RE white reflex. At 7 weeks he was examined by an ophthalmologist and on the following day developed red eye. Up on referral to us at 9 weeks of age, his RE was fully field by the tumor mass with areas of extra ocular extension and orbital inflammation that simulated cellulites. On examination of the LE an exophytic white tumor was detected, measuring 13X14 mm in base 7.6 mm in high associated with serous retinal detachment. MRI Study demonstrated enhancement of the pineal body. The patient was diagnosed as TRb and was treated with the Children Oncology Group (COG) protocol ARET0321 designed for children diagnosed with extra-‐

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ocular retinoblastoma. He received 4 cycles of Vincristine, Cisplatin, Cyclophosphamide and Etoposide. A peripheral stem cell harvestman was not possible due to his young age. Cyotherapy and Trans papillary thermo therapy were used for consolidation. Results: The RE was successfully enucleated after completion of chemotherapy and marked regression on MRI. No viable tumor cells were seen in the specimen. The optic nerve end was not infiltrated. The tumor in the LE regressed to a calcified mass measuring 3.77 mm in base 1.26 mm in high with no serous retinal detachment. No enlargement of the pineal or leptomeningeal involvements was seen on follow-‐up MRI. The patient is stable with no recurrence in 7 months follow-‐up. Conclusions: Oncology Group (COG) protocol ARET0321 could be used for the treatment of young patients with TRb .Long term follow up is needed to role out late ocular or brain recurrence. 9:42 -‐ CONSERVATIVE TREATMENT OF ADVANCED BILATERAL RETINOBLASTOMA: COMPARISON OF 1995-‐2003 TO 2004-‐2009. LIVIA LUMBROSO-‐LE ROUIC; ALEXIA SAVIGNONI; ISABELLE AERTS; CHRISTINE LEVY-‐GABRIEL; LAURENCE DESJARDINS, Institut Curie, Paris, France Introduction: The aim of the study is to show the evolution of and improvement of conservative treatment protocols for advanced (group D) retinoblastoma compared to the less advanced stages. Materials & Methods: Retrospective study on all patients with bilateral retinoblastoma treated in our institution during the study period (1995-‐2009). Before 2003 all patients were treated by two courses of VP16 and carboplatin followed by chemothermotherapy and other ocular treatments such as cryo and plaques, or external beam radiation. From 2004 on, the management was modified, the patients with bilateral group D eyes (or on the only one amenable to a conservative treatment) received 6 courses of 3 drugs (VP16, carboplatin and vincristine) with intensive local treatment (laser thermotherapy and cryo on the tumor sites at each chemotherapy cycle from the third course). Data concerning initial staging, treatments and results were registered in the data base. A comparison between the two groups is realized regarding tumor control and ocular preservation without EBR. Results: There were 317 children with bilateral retinoblastoma during the study period. Conservative management was performed for at least one eye in 308 children and for both eyes in 108 children, (425 eyes conservatively treated). There were 21% group A, 43% group B, 12% group C, 22 % group D and 2% group E. The median follow up is 72 months. Median age at diagnosis was 7 months. During the first period (1995-‐2003) 25 % of conservative management required external beam radiation essentially for the group D eyes, while only 10% during the second period (p 0.0000243) Secondary enucleation (after the first year) was 8% during the first period versus 3% during the second period. Conclusions: Adaptation of the chemotherapy protocol to the initial staging improves eye retention without external beam. Local ocular treatments such as cryo and laser thermotherapy associated to the chemotherapy protocol improve tumor control.

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09:55 -‐ 10:40 Ellsworth Lecture: "Seeding in retinoblastoma:

recognition, management, and regression patterns"

Dr Francis MUNIER, Lausanne, Switzerland-‐ Introduced by Dr Brenda GALLIE


11:30 -‐ 12:54 Retinoblastoma Session 3 Treatment & Miscellaneous

Chairs: Francis MUNIER & Hossam EL-‐ZOMOR

11:30 -‐ INTRAVITREAL MELPHALAN FOR RETINOBLASTOMA WITH CLOUDY VITREOUS SEEDS. MARIE-‐CLAIRE GAILLARD; AUBIN BALMER; STEFANO BINAGHI; MAJA BECK-‐POPOVIC; FRANCIS L MUNIER, Jules Gonin Eye Hospital, Lausanne, Switzerland Introduction: Vitreous seeds represent the major cause of failure and consequent enucleation in the treatment of retinoblastoma (Rb). High-‐density vitreous seeds may take on an aspect of « clouds », these masking the primary tumor and constituting one of the most challenging clinical situations in Rb treatment. The 50% failure rate associated with systemic chemotherapy due to poor penetration of the chemotherapeutic agent in the vitreous was already improved to 30% with the advent of intra-‐arterial chemotherapy. We report the preliminary results of this severe form of seedings treated by intravitreal injections. Materials & Methods: 10 eyes with vitreous seeds in the form of « clouds », diffuse (N=9) or localized (N=1), were treated with 3 to 10 intravitreal injections of melphalan, concomitant with primary tumor treatment (N= 6 focal, N=2 intra-‐arterial chemotherapy, N= 2 systemic chemotherapy). An anterior chamber puncture of aqueous liquid is performed to prevent vitreal reflux of fluid contaminated with potentially malignant cells. The sample is sent for cytopathologic analysis. The injection site is assessed by UBM to ensure the absence of exclusion criteria such anterior or posterior chamber invasion, anterior hyaloid or retinal detachment, or presence of tumor at the entry site. 20 to 38 µg melphalan are injected with a 32 G needle. Upon needle removal three cycles of freeze and thaw cryocoagulation are applied to sterilize the needle track. Results: Vitreous seeds regressed in all cases from 1 to 16 months after the first injection. There were no systemic or ocular complications apart from salt-‐and-‐pepper retinopathy localized at the injection site. No relapse of vitreous seedings was observed after a mean follow-‐up of 14 months. Conclusions: Intravitreal injections increase the concentration of chemotherapeutic agents, optimizing the response in the cloudy, resistant form of vitreous seeding. The risk of extra-‐ocular spread is real in these high-‐density tumor filled globes. For this reason, strict security control of the injection site by UBM and an adapted injection technique to ensure the safety of the procedure are mandatory in these cases.

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11:42 -‐ INTRA-‐ARTERIAL CHEMOTHERAPY USING MULTI AGENT CHEMOTHERAPY (THREE DRUGS) FOR TREATMENT OF RECALCITRANT INTRAOCULAR RETINOBLASTOMA (EXTENDED FOLLOW UP). BRIAN MARR; Y. PIERRE GOBIN; IRA J. DUNKEL; SCOTT E. BRODIE; DAVID H. ABRAMSON, Memorial Sloan Kettering Cancer Centre & Weill Cornell Medical College, New York, N.Y Introduction:Throughout modern oncology the use of single agent chemotherapy for successful treatment of solid cancers is unheard of, barring a few exceptions, one of these being Intraarterial chemotherapy (IAC) for treatment of retinoblastoma. We report our experience with IAC using simultaneous multi agent chemotherapy (three drugs) Materials & Methods: A retrospective chart review of 26 eyes of 25 patients receiving three-‐drug (melphalan, topotecan, and carboplatin) multi agent IAC was conducted between May 2006 and June 2011. Results: Twenty-‐six eyes, Reese-‐Ellsworth group 5b (22) 5a (1) 4a(2) and 3a(1), received 61 infusions of 3 drug multiagent chemotherapy for treatment of advanced retinoblastoma. The median number of infusions was 2 with a maximum of 4 and minimum of 1 averaging 2.3 per eye. Fourteen of 25(56%) patients presented after failing intravenous chemotherapy (IVC), 2/25(8%) after failing IVC and external beam radiotherapy and 1/25(4%) after failing IVC and plaque brachytherapy. 19 /26 (73)% of eyes were salvage over a mean follow up period of 34 mo. (24-‐67 mo.) Conclusions: We have now successfully used a three-‐drug multi-‐agent IAC in an attempt to save eyes that have failed IVC and single or double agent IAC and the results have been durable. 11:54 -‐ HIGH RISK HISTOPATHOLOGIC FEATURES OF RETINOBLASTOMA IN EGYPT; TREATMENT OUTCOMES. HOSSAM ELZOMOR; ADEL ALEIELDIN; HALA TAHA; RADWA NOUR; AHMAD S ALFAAR, Children's Cancer Hospital Egypt 57357. -‐ National Cancer Institute, Cairo, Egypt Introduction: Retinoblastoma is the most common intraocular malignancy of infancy and early childhood. In developing countries the decreased access to health care which poses a major challenge and increases the possibility of extra-‐ ocular dissemination. Surgical pathological examination aim at discerning histopathological features that pose a risk for disease dissemination and are indicative of a need for adjuvant therapy. Our aim is to study the prevalence of Histopathological high risk features amongst the enucleated eyes, measure the overall survival in those patients and compare two groups 1) the ones who received adjuvant chemotherapy, 2) the ones who did not. Materials & Methods: We retrospectively analyzed patients who were presented to Children’s Cancer Hospital -‐ Egypt 57357 between July 2007 and December 2012. Only unilateral cases were included in the study. Pathological examination of the enucleated eyes was conducted for all cases. Pathologists were trained for detection of high risk features. Designated high risk features included massive choroidal invasion, retrolaminar invasion or any degree of combined choroidal with optic nerve invasion. Follow-‐up was conducted to June 2013.

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Results: During the period from July 2007 to end of Dec 2012 a total of 262 patients presented to CCHE-‐57357 with retinoblastoma. Only 40 patients had unilateral retinoblastoma and undergone enucleation revealing high risk features.Three patients have been diagnosed later with extra-‐ocular retinoblastoma. Only %7.75 of patients with unilateral retinoblastoma and high risk features developed metastatic disease. Conclusions: Introducing adjuvant chemotherapy for our cases has improved event free survival. Only followup with no adjuvant treatment is sufficient for patients with no high risk features. Training pathologists in developing countries on detection of high risk features is a cost-‐effective measure for improving retinoblastoma integrated management. 12:06 -‐ PATHOLOGICAL FINDINGS OF RETINOBLASTOMA IN EGYPT; IMPLEMENTING CAP PROTOCOL IN DEVELOPING COUNTRIES. HALA TAHA; HOSSAM ELZOMOR, ALAA ELHADDAD; ADEL ALEIELDIN; M. SAAD ZAGHLOUL; RADWA NOUR; AHMAD S. ALFAAR, Children's Cancer Hospital -‐ Egypt 57357. -‐ National Cancer Institute, Cairo, Egypt Introduction: Retinoblastoma is the most common intra-‐ocular tumour of childhood. Radiotherapy and chemotherapy have improved survival for the disease over the last 40 years. Looking for prognostic criteria is one of the major research areas that inspires disease management teams allover the world. Many protocols were designed over the years to correlate the presence of certain pathological and/or molecular features and prognosis of the disease. Childrenâ?Ts Cancer Hospital Egypt 57357 (CCHE) is a new cancer center in Egypt that serves about 25-‐40% of the pediatric oncology population in the country. The aim of this study is to present the histopathological findings in Egypt leading pediatric oncology center and impact of implementing CAP protocol on the improvement of patient care. Materials & Methods: CCHE Retinoblastoma study team implemented College of American Pathologist (CAP) protocol of Retinoblastoma version 3.0 (Based on AJCC/UICC TNM, 7th edition) for examining enucleation specimens. Pathology team has conducted an internal training for pathologists. New electronic case report forms (eCRFs) were designed for reporting the progress. Real-‐time online statistical analysis system was integrated with eCRFs. Implementation included prospective processing and data entry of new specimens properties beside revising of all specimens prior to the implementation of the protocol. Healthcare quality measures included required time for releasing the reports. Results: Between July 2007 and May 2013 we’ve examined 198 enucleation specimens. Mean of all ocular diameters was 22.2 mm. Mean Optic nerve stump length was 6.35 mm. Invaded structures included Cornea (5.3%), Anterior chamber (15.4%), Iris (8.3%), Lens (5.9%), Ciliary body (18.3%), Choroid, (72.2%), Sclera (17.2%), and Orbital tissue (7.1%). Any degree of Optic Nerve invasion was noted in 51% of specimens while surgical margin was involved in 10.8%. Validation rules in eCRFs helped the team to complete missing data and provide systematic reporting. Integrated online analysis system provided a real-‐time reporting facility for detecting protocol implementation progress.

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Conclusions: Implementing CAP retinoblastoma protocol resembled a roadmap for improving patient care. It supported the systematic reporting of cases, decreasing errors and missing data and provided research informatics team with a blueprint for developing a real-‐time monitoring system. This system can be integrated in the practice of emerging teams to ensure adherence to quality measures. 12:18 -‐ SODIUM IODIDE SYMPORTER IS DIFFERENTIALLY EXPRESSED IN RETINOBLASTOMA AND CORRELATES WITH THE AGGRESSIVENESS OF TUMORS. J. SAMUEL; R. K. KANWAR; J. R. KANWAR; S. KRISHNAKUMAR. Department of Nanobiotechnology Vision Research Foundation, Sankara Nethralaya, Chennai, India. (ISGEDR TRAVEL AWARD RECIPIENT) Purpose: In thyroid cancer, radiotherapy is sensitive because of Sodium Iodide Symporter (NIS) and it is exploited for diagnostic imaging and molecular radionuclide-‐based therapy. The understanding of the biology of NIS has been advancing rapidly in the last two decades and the presence of NIS in radiosensitive retinoblastoma (RB) is presently unknown. The present study was aimed to evaluate (i) the NIS protein expression in RB tumors and (ii) whether NIS immunoreactivity correlates with tumor aggressiveness. Methods: NIS protein expression was analyzed in advanced stage tumor samples (Grade D,E) from the RB patients’ by immunohistochemistry, western blotting, flow cytometry and also compared with established standard RB cell line (Y79) by immunofluorescence. Results: NIS protein expression was observed in 20/20 (100%) tumor samples and found to be associated with the clinical features of the tumors. Immunohistochemistry and flow cytometric analysis (FACS) revealed that NIS protein expression was extensively higher in the RB cases of choroid and/or optic nerve invasion when compared to non-‐invasive tumors (P<0.05). However, the adjacent normal retina cells did not show any NIS expression. Immunofluorescence revealed strong membrane positivity in RB cell line. Western blotting revealed strong partially glycosylated protein at ~75kDa, ~50kDa and a faint underglycosylated protein at ~25kDa. Conclusion: In conclusion, using both clinical RB tumor samples and established RB cell line Y79, our study to the best of our knowledge, demonstrates for the first time the presence of NIS protein in RB and its correlation with the aggressiveness of tumors. Further detailed investigations of NIS molecule at RNA and protein levels, its functionality and the fate of transported iodine are required to understand the role of NIS in RB tumor biology. In addition, the mechanistic importance of NIS in diagnosis, treatment and monitoring of RB patients can be done by employing more number of patients using different tumor types. 12:30 -‐ IDENTIFICATION OF MOST COMMONLY USED WEB-‐BASED SEARCH TERMS FOR LEUKOCORIA. SANDRA E STAFFIERI; ALEX HEWITT; LISA KEARNS; DAVID MACKEY, Centre for Eye Research Australia, University of Melbourne, Royal Victorian Eye & Ear Hospital, East Melbourne, AUSTRALIA

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Introduction: Leukocoria is the most common presenting sign for retinoblastoma and cataract. Delayed diagnosis for retinoblastoma is common and can result in further advancement of the disease with adverse outcomes â?” enucleation, spread along the optic nerve, systemic spread or death. Delayed diagnosis in cataract can lead to severe visual impairment. Often the leukocoria is seen by the parent in family photographs or with the naked eye. The explosion of ready access to health information on the internet is well recognised, and increasing at a rapid rate. The Pew Internet and American Life Project found 77% of individuals turning to the internet to seek health related information do so through search engines. This project aims to identify the free text search words parents might use in search engines to seek further information on their photographic findings. By determining the most commonly used free text search words, search engine optimisation can be utilised to direct internet users to reliable information regarding retinoblastoma and cataract. This would include the importance of seeking urgent referral to a medical practitioner when leukocoria is seen in a photograph. Better access may in turn lead to reduced delays in diagnosis and improved outcomes for the affected individual. By virtue of participation in the interactive questionnaire an additional outcome of this project will be the increased awareness of the most common presenting sign for retinoblastoma -‐ leukocoria. Materials & Methods: Two photographs depicting leukocoria in a child with retinoblastoma were incorporated into an interactive website questionnaire. The website includes the ability to monitor search terms used by the participant to seek further information about leukocoria as seen in the photographs, as well as identify the public perception of the need to obtain an urgent medical opinion. Results: The interactive website in English has been completed and recently launched on parenting websites and magazines, maternal and child health nurse clinics and with community groups. The website will be demonstrated and preliminary data discussed. Conclusions: We will present an interactive website to determine general public responses to leukocoria as seen in photographs, and identify the most common free text search words used on web-‐based search engines to seek further information. Collaboration is invited to undertake this project internationally. 12:42 -‐ HIGH RESOLUTION SNP ARRAY PROFILING IDENTIFIES VARIABILITY IN RETINOBLASTOMA GENOME STABILITY. BERBER MOL; CHARLOTTE DOMMERING; ANNETTE MOLL; JACQUELINE CLOOS; JOSEPHINE DORSMAN, VU University Medical Center, Amsterdam, The Netherlands Introduction: Both hereditary and non-‐hereditary retinoblastoma (Rb) are commonly initiated by loss of both copies of the retinoblastoma tumour suppressor gene (RB1), while additional genomic changes are required for tumour initiation and progression. Clinically there are important differences between hereditary and non-‐hereditary Rb patients, such as age at diagnosis, bilateral or unilateral disease, risk for development of secondary tumours and unifocal or multifocal disease. Our purpose was to determine whether there is genomic heterogeneity between different clinical Rb subtypes. Materials & Methods: Twenty-‐one Rb tumours from 11 hereditary patients and 10 non-‐hereditary Rb patients were analysed using high-‐resolution Single Nucleotide

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Polymorphism (SNP) arrays. Copy number changes and loss of heterozygosity were determined and gene losses and gains were validated with Multiplex Ligation-‐dependent Probe Amplification. Results: There was a large difference in number of copy number aberrations between tumours. A subset of non-‐hereditary Rbs turned out to be the most genomic unstable, while especially very young patients with hereditary Rb display stable genomes. Established Rb copy number aberrations, including gain of chromosome 1q and loss of chromosome 16q, turned out to be preferentially associated with the non-‐hereditary Rbs with later age of diagnosis. In contrast, copy number neutral loss of heterozygosity was detected mainly on chromosome 13, where RB1 resides, irrespective of hereditary status or age at diagnosis. Focal amplifications and deletions and copy number neutral loss of heterozygosity besides chromosome 13 appeared to be rare events in Rb. Conclusions: We showed that there are differences in genome stability between Rb tumors, with tumors from hereditary patients with young age at diagnosis being most stable and tumors from non-‐hereditary patients with old age at diagnosis being most unstable. Understanding of the genomic heterogeneity of Rb tumours can offer perspective for the development of novel therapy and individualized treatment. 12:54 -‐ 14:00 Lunch -‐ Provided on Site

Poster Viewing

14:00 -‐ 14:54 Genetics Session 6 Clinical & Diagnostic Aspects

Chairs: Birgit LORENZ & Arif O KHAN

14:00 -‐ TUBEROUS SCLEROSIS -‐ WHY WE NEED AN OPHTHALMIC CHECK-‐UP. CHRISTINA GERTH-‐KAHLERT: GABRIELE WOHLRAB; SANDRA TALLE-‐STUCKI; KLARA LANDAU, University Hospital Zurich, Zurich, Switzerland Introduction: Retinal manifestation in Tuberous sclerosis complex (TSC) is one of the main diagnostic criteria for establishing the clinical diagnosis. Retinal astrocytic harmatoma are observed on up to 36% of examined patients (Aronow et al. 2012). Once diagnosed, do we need to follow patients with TSC in our ophthalmic care? Materials & Methods: Patients diagnosed with TSC were identified through the Zurich TSC network. Patients received a comprehensive and dilated eye examination including photographic documentation and, if possible optical coherence tomography (OCT). Results: Ophthalmic records were available in 15 of 44 patients known to have TSC (12 female, 3 male; ages 2 weeks to 44 years). In 10/15 patients retinal astrocytic harmatoma were described. Complications were seen in 3 patients: (1) optic atrophy secondary to papilloedema associated with increased intracranial pressure in subependymal giant cell astrocytoma (SEGA) and (2) retinal nerve fiber layer (RNFL) atrophy due to vigabatrin therapy of infantile epilepsia in 2 patients. Conclusions: Once diagnosed, patients with TSC are required to have a regular ophthalmic exam:

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(1) to follow and, if necessary, support visual development in affected children (2) to rule out increased intracranial pressure especially in developmental delayed and handicapped patients (3) to monitor for treatment side effects. 14:12 -‐ COLOUR VISION IN STARGARDT DISEASE. TINE VANDENBROUCKE; COULOUR VISION in STD STUDY GROUP; ELFRIDE DE BAERE; BART P. LEROY, Ghent University Hospital, Ghent, Belgium Introduction: To investigate the type and severity of colour vision deficiencies (CVDs) in Stargardt disease (STD) and to establish whether there is a relationship between either best-‐corrected visual acuity (BCVA) or full field ERG (ffERG) and degree of CVD. Materials & Methods: A retrospective, cross-‐sectional study of 73 patients with a molecularly proven diagnosis of STD, who underwent extensive colour vision testing using a battery of colour vision (CV) tests. In addition, thirteen patients underwent anomaloscopy. Results: Patients were allocated to 4 BCVA groups and to 3 ffERG groups. Normal colour vision was found in almost 20% of patients. R/G CVDs increased as BCVA declined. About 45% of all R/G CVDs were of the deutan type of CVD, although protan R/G CVDs became progressively apparent as BCVA decreased. A predominance of pseudoprotanomaly was evident only on anomaloscopy. Additional Blue/Yellow (B/Y) CVDs were noted in about 25% of patients. B/Y CVDs and BCVA better than 0.20 LogMar acuity were seen in adult-‐onset STD. CVDs evolve to scotopization in patients with low BCVA and/or longstanding disease. Also, no statistically significant differences in ERG results were found between groups with or without a CVD. Conclusions: Colour vision function is better correlated to BCVA than to ffERG results in STD. The presence of CVDs may help to establish an early diagnosis of STD, especially in cases of adult-‐onset disease. A battery of CV tests is required to reliably diagnose a CVD. 14:24 -‐ THE PHENOTYPIC VARIABILITY OF RETINAL DYSTROPHIES ASSOCIATED WITH MUTATIONS IN CRX. SARAH HULL, Moorfields Eye Hospital, London, UK 14:36 -‐ PATHOGNOMONIC RETINAL DYSFUNCTION AND DYSTROPHY IN CHILDREN. ARIF O. KHAN, King Khaled Eye Specialist Hospital, Jeddah, Kingdom of Saudi Arabia. Introduction: Most childhood retinal dysfunctions and dystrophies are not specific, are collectively labeled as “retinal dystrophy,” and can be caused by mutations in many different genes. However, some clinical phenotypes are distinct and specific for particular gene mutation. This presentation highlights features phenotypes of retinal dysfunctions and dystrophies in children that are pathognomonic and specific for particular gene mutations. Materials & Methods: Retrospective case series. Results: All cases clinically diagnosed with congenital cone-‐rod synaptic disorder (11 children from 4 families), cone dystrophy with supranormal rod responses (9 children from 7 families), and the vitreoretinopathy of Knobloch syndrome (9 children from 6 families) were found to have mutations in the specific gene associated with the

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respective condition. Three of 4 children (from 4 families) clinically diagnosed with enhanced S-‐cone syndrome had mutation in a gene commonly associated with the condition. Conclusions: The childhood clinical features of congenital cone-‐rod synaptic disorder, cone dystrophy with supranormal rod response, Knobloch syndrome, and enhanced S-‐cone syndrome are distinct. These conditions should be recognized rather than lumped as “retinal dystrophy.” 14:48 -‐ Phenotype of RDH12-‐related Early-‐Onset Retinal Dystrophy. JULIE DE ZAEYTIJD, THE RDH12 STUDY GROUP (F Coppieters, L Visser, FL Munier, S Walraedt, I Casteels, I Perrault, JM Rozet, J Kaplan, DF Schorderet, T de Ravel, RWJ , Collin, E De Baere, C Hamel, LI van den Born), BART LEROY, Ghent University Hospital, Ghent, Belgium Introduction: To describe the phenotype in Early-‐Onset Retinal Dystrophy (EORD) related to RDH12 mutations. Materials & Methods: Twenty-‐three affected individuals from fourteen families with proven RDH12 mutations underwent a detailed ophthalmological examination including fundus photography using white, autofluorescent, near-‐infrared and red-‐free light and optical coherence tomography (OCT). In addition, psychophysical and lectrophysiological testing (ISCEV-‐standard ERG) was performed. Results: All twenty-‐three affected individuals had a history of poor vision from the first few years of life. Fundoscopy showed marked atrophy and yellow discolouration of the macula. Spicular intraretinal pigmentation was present in the (mid) periphery of all fundi. In addition, a remarkable aspect of patchy preservation of functional retina in the retinal periphery was present until relatively late in the disease with additional significant sparing of the peripapillary area in all individuals. OCT confirmed the conservation of the peripapillary retinal structure. ERG revealed very reduced to absent responses under both scotopic and photopic conditions. Conclusions: The phenotype of RDH12-‐related EORD includes an early macular atrophy with yellow discolouration, and patchy preservation of peripheral and peripapillary retina as a specific pathognomonic feature. 15:00 -‐ 16:30 Symposium

How will increasingly efficient genetic diagnostics impact your patients? What needs to happen to take advantage of genetics? How are you using genetics in patient care now, and in 3 years? How will you determine the most cost-‐effective approach (timing, genes tested, clinical practice)?

Chairs: Elise HEON & Edwin M STONE

15:00 Introduction to symposium & my experience,

views, ideas & queries Elise HEON Toronto, Canada

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15:05 My experience, views, ideas & queries Isabelle AUDO Paris, France

15:10 My experience, views, ideas & queries Andrew WEBSTER and Anthony MOORE London, UK

15:15 My experience, views, ideas & queries Arlene DRACK Iowa City, IA, USA

15:20 My experience, views, ideas & queries Helen DIMARAS Toronto, Canada

15:25 My experience, views, ideas & queries Bart P LEROY Ghent, Belgium & Philadelphia, PA, USA

15:30 – 16:10 Panel discussion E HEON,E STONE, I AUDO, A MOORE/A WEBSTER, A DRACK, H DIMARAS, B GALLIE, BP LEROY

16:10 QUESTIONS PANELISTS

16:25 Chairperson Summary Elise HEON Toronto, Canada


17:00 -‐ 18:30 Belgian RP Patients Session

Association Retina Pigmentosa -‐ Ophthalmic Genetics

Chairs: Viviane HALLET-‐TORDEURS & Bart P LEROY

17:00 Introduction Bart P LEROY

Ghent, Belgium & Philadelphia, PA, USA

17:05 Basics of Genetics Elias I TRABOULSI Cleveland, OH, USA

17:25 Update on Genetic Testing Edwin M STONE Iowa City, IA, USA

17:45 Using Genetic Tests in the Clinic Elise HEON Toronto, Canada

18:05 Update on Gene Therapy Jean BENNETT Philadelphia, PA, USA

18:25 Conclusions Bart P LEROY, Ghent Belgium & Philadelphia, PA, USA

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19:30 -‐ ON Gala Dinner All Registered Participants

Hotel Falligan

Kouter, 172, 9000 Ghent

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POSTERS

Board # Title 1st author City, Country 1 A case of late presentation of

retinoblastoma Cristina SANTOS Amadora,

Portugal 2 Retinoblastoma, a review of

cases Inês COUTINHO Amadora,

Portugal 3 Knowledge of retinoblastoma

genetics in Canadian adult retinoblastoma survivors

Helen DIMARAS Toronto, Canada

4 Presentation of extra-‐ocular retinoblastoma in Egypt

Hossam EL-‐ZOMOR Cairo, Egypt

5 Expression of ABCG2, MCM2, embryonic stem cell markers and VEGF in retinoblastoma

Bhavna CHAWLA New Delhi, India

6 Subretinal gene therapy in Bardet-‐Biedl syndrome type I mice

Arlene DRACK Iowa City IA, USA

7 Exome sequencing identifies RDH12 compound heterozygous mutations in a family with severe retinitis pigmentosa

Juan Carlos ZENTENO Mexico City , Mexico

8 Homozygosity mapping identifies the CLN3 gene as responsible for a recessive form of retinitis pigmentosa

Beatriz BUENTELLO-‐VOLANTE

Mexico City , Mexico

9 Novel and known FRMD7 mutations in Belgian patients with X-‐linked idiopathic infantile nystagmus

Basamat AL-‐MOALLEM

Ghent, Belgium

10 Exome sequencing identifies a novel RP1 mutation in a Belgian family with autosomal dominant retinitis pigmentosa

Caroline VAN CAUWENBERGH

Ghent, Belgium

11 Variable phenotype in Enhanced S-‐Cone Syndrome

Barbara JANSSENS Ghent, Belgium

12 Ocular phenotype of Jalili syndrome

Sofie GEENS Ghent, Belgium

13 Ocular development and axial length in the bestophinopathies

Julie DE ZAEYTIJD Ghent, Belgium

14 Unilateral X-‐linked retinoschisis Michael RITZ Strasbourg, France

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15 Retinal histopathology in eyes from patients with autosomal recessive retinitis pigmentosa caused by eys mutations

Gayle PAUER Cleveland, USA

16 Genetic counselling remains challenging in Leber's optic neuropathy

Valérie PELLETIER Strasbourg, France

17 Craniofrontonasal syndrome in a male with sagittal craniosynostosis

Jacqueline HOOVER Pittsburgh, PA, USA

18 Ocular features of microcephaly with or without chorioretinopathy, lymphedema, or mental retardation (MCLMR) syndrome due to KIF11 mutations

Irina BALIKOVA Brussels, Belgium

Board #1 -‐ A CASE OF LATE PRESENTATION OF RETINOBLASTOMA. CRISTINA SANTOS; INÊS COUTINHO; CATARINA PEDROSA; MÁRIO RAMALHO; SUSANA TEIXEIRA, Hospital Prof. Doutor Fernando Fonseca, EPE, Amadora, Portugal Introduction: Retinoblastoma is the most frequent intraocular tumor in children. Mean age at diagnosis for unilateral disease is at 24 months and for bilateral disease at 13 months. More than 90% of the cases are diagnosed before 5 years of age. Materials & Methods: The purpose of this poster is to describe a case of retinoblastoma diagnosed at the age of 9 years of age. The case is well documented by anamnesis, fundoscopy, retinography, fluorescein angiography, ultrasound, magnetic ressonance and peripheral blood genetic testing. Results: Healthy 9 years old girl with one month history of leukocoria and decreased vision in her right eye (OD) is referred to our hospital for diagnostic counselling. Best corrected visual acuity in her right eye was 20/200 and 20/20 in her left eye. Slit lamp examination was unremarkable except for dense anterior vitreous infiltrates. Fundoscopy revealed a retrolental subretinal yellow mass, partial retinal detachment, dilated tortuous retinal vessels and extensive vitreous seeding. Ultrasound revealed a 9.4mm exophytic mass with calcification. UBM excluded ciliary body involvement and magnetic ressonance excluded extrascleral spreading and optic nerve involvment. Presumptive stage D retinoblastoma diagnosis was established. Since the only conservative treatment strategies available at our Center are systemic chemotherapy, cryotherapy and LASER photocoagulation, the Pediatric Oncology department at Hôpital Ophthalmique Jules Gonin was contacted and promptly available to treat the child.

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Intraarterial melphalan chemotherapy was performed, with tumor size reduction after one cycle. Intravitreal melphalan injection was not safe after this first chemotherapy cycle since there was evidence of vitreous seeding in all four quadrants at the level of pars plana. Further two intraarterial chemotherapy cycles are planned. Conclusions: This case report ilustrates a late presentation of retinoblastoma characterized by extensive vitreous seeding. Board #2 -‐ RETINOBLASTOMA -‐ A REVIEW OF CASES. INES COUTINHO; CRISTINA SANTOS; MARIO RAMALHO; JOÃ£O CABRAL; SUSANA TEIXEIRA, Hospital Prof.Doutor Fernando da Fonseca Introduction: Retinoblastoma is the most common malignant ocular tumor of childhood with an incidence of 1: 15000 â?" 20000 births and it is due to a mutation on RB1 gene, a tumor supressor gene. The average age of diagnosis is 12 months in bilateral and 24 months in unilateral cases. Late diagnosis usually means loss loss of eye function or even death, that’s why the early screening in every child is so critical. The goal is review/approach the diagnosis and current treatment of retinoblastoma and describe the experience in treatment and follow-‐up of the children in our hospital, a Portuguese not central hospital near Lisbon. Materials & Methods: Review of the literature. Retrospective evaluation of the patients treated in the last 8 years in our hospital, together with Portuguese institute of oncology using local (lasertherapy, cryotherapy and enucleation) and systemic (chemotherapy) therapy. Results: 7 patients were treated with unilateral retinoblastoma and 4 with bilateral disease. They were send to us by the assistant physician (pediatrician, ophthalmologist and general practice physician) because a positive familiar history, emerge of leukocoria or strabismus. The average age was 21 months and the most frequent group was D according international classification system for intraocular retinoblastoma In the 8 eyes where conservative treatment was possible, after chemoreduction they were submitted to LASER Argon photocoagulation or cryotherapy, according to the tumor location. In five of these eyes treatment with transpupilar thermotherapy (TTT), brachytherapy, intra-‐vitreous or intra -‐arterial chemotherapy, in Laussanne, at HÃ´pital ophtalmique Jules-‐Gonin was also performed. 4 eyes were first enucleated and another 3 were secondary enucleated. The authors exhibit images taken before, during and after treatment Conclusions: Leukocoria and strabismus are the main forms of presentation of retinoblastoma. The treatment depends of the size of the tumor, laterality, eyesight potential and age of the child. The main therapeutic approaches are systemic chemotherapy and local therapies like cryotherapy, laser therapy, intra-‐arterial and intravitreous chemotherapy, brachytherapy and enucleation. Earlier diagnosis and treatment increase the number of patients that remain free of disease and preserve the vision.

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Board # 3 -‐ KNOWLEDGE OF RETINOBLASTOMA GENETICS IN CANADIAN ADULT RETINOBLASTOMA SURVIVORS. DIMARAS H; HE B. University Of Toronto, Department Of Ophthalmology & Visual Sciences, Toronto, Canada Introduction: To assess knowledge of Canadian adult retinoblastoma survivors about retinoblastoma genetics, associated inheritance patterns and second cancer risks. Materials & Methods: Cross sectional-‐survey design. Survivors were recruited by e-‐mail or in person with the assistance of the Canadian Retinoblastoma Society and Canadian retinoblastoma specialists. Survivors were asked to complete an on-‐line questionnaire that inquired about their demographics and retinoblastoma history, and assessed their knowledge about retinoblastoma genetics, inheritance patterns and second cancer risks. Results: Fourteen adult retinoblastoma survivors (50% female; 50% bilateral) participated in the survey (mean age 37, range 19-‐74 years). The mean knowledge score on the study questionnaire was 67% (StDv: 20%; 95% CI: 55%~78%; min: 24%, and max: 91%). Most survivors were aware of the major long-‐term health risks related to their primary retinoblastoma diagnosis, and many survivors could correctly identify strategies to cope with/reduce associated second cancer risks. Our cohort was found to communicate extensively with their doctors: 93% reported obtaining retinoblastoma information from their ophthalmologist, oncologist and/or genetic counselor; 43% reported obtaining additional retinoblastoma information from their parents. Survivors who performed well below average (3/14) were found to have had limited or no follow-‐up with a genetic counselor or other specialist after the age of 18. While no demographic (sex, age etc.) or disease-‐history (laterality, treatment course, etc.) trait was significantly associated with knowledge score, those with offspring or relatives with retinoblastoma showed a trend towards higher knowledge scores. Conclusions: Canadian adult survivors of retinoblastoma have a reasonable understanding of retinoblastoma genetics, inheritance patterns and second cancer risks, particularly when they participate in long-‐term follow-‐up care. Our results inform long-‐term genetic follow-‐up care for retinoblastoma survivors in Canada. Board #4 -‐ PRESENTATION OF EXTRA-‐OCULAR RETINOBLASTOMA IN EGYPT. HOSSAM ELZOMOR; RADWA NOUR; AHMAD S ALFAAR; ADEL ALEIELDIN; M SAAD ZAGHLOUL; HALA TAHA; ALAA ELHADDAD, Children's Cancer Hospital, National Cancer Institute, Cairo, Egypt Introduction: Extraocular Retinoblastoma is a disease of developing countries. It represents a challenge for oncologists with its poor prognosis and resistance to chemotherapy. There is a major discrepancy in reporting its incidence between developing and developed world. Egypt represents the third most populous African country, hence, establishing Childrenâ?Ts Cancer Hospital Egypt 57357 (CCHE) -‐a non for profit institute-‐ represents a hope for childhood cancer patients in Egypt and the middle-‐east. The aim of this paper is to report the magnitude of the disease and presenting features of extraocular retinoblastoma in Egypt over the last 6 years. Materials & Methods: We reviewed all the cases that were presented with extraocular retinoblastoma to our center between July 2007 and December 2012. This included

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reviewing clinical, radiological and pathological investigations. Cases were classified based on international staging of Retinoblastoma and milestone of diagnosing the disease. Results: Between July 2007 and December 2012 our team has diagnosed 48 cases of metastatic disease accounting for 18.3% of all cases with retinoblastoma. Mean age of presentation was 27.3 months and duration of prior symptoms was 17 months. 40% of the cases presented initially with extra-‐ocular retinoblastoma while 49% was diagnosed after enucleation and about 9% of the cases ignored followup sessions and came later with metastases. Stage II represented 32.4% of the cases while Stage III was 26.4% and Stage IV accounted for 42.2 % of the cases. Conclusions: Extraocular retinoblastoma is still an under-‐studied field due to the few diagnosed cases in developed world. But in the settings of developing countries it represents a real nightmare specially with the increasing number of advanced intra-‐ocular retinoblastoma patients and lack of access to healthcare services. Board #5 -‐ EXPRESSION OF ABCG2, MCM2, EMBRYONIC STEM CELL MARKERS AND VEGF IN RETINOBLASTOMA. BHAVNA CHAWLA; SUJATA MOHANTY; MANISHA SINGH; RENU SAINI; RAJVARDHAN AZAD, All India Institute of Medical Sciences, New Delhi, India Introduction: Retinoblastoma is the most common intraocular malignancy in the pediatric age group. Cancer stem cells (CSC) are believed to be related to aggressiveness and chemoresistance of the tumor. VEGF is an important factor which is associated with tumor spread by angiogenesis. The present study was undertaken to determine the expression of ABCG2, McM2, Embryonic Stem cell markers (Nanog & Oct-‐4) and VEGF in human retinoblastoma tissue. Materials & Methods: Fresh unfixed retinoblastoma tissue was harvested from the enucleated eyeball of children who underwent primary enucleation for retinoblastoma. The tumor samples were transported to the laboratory and were analysed for the presence of GAPDH, ABCG2 & McM2, Nanog & Oct-‐4, and VEGF by reverse transcriptase-‐ polymerase chain reaction (RT-‐ PCR) testing. Results: A total of 55 cases were studied. Two samples were found to be negative for GAPDH and the remaining 53 samples underwent further analysis. RT-‐ PCR results showed that Nanog & Oct-‐4 were expressed in 75.5% cases, while McM2 and ABCG2 in 54.7% cases. VEGF was positive in 69.8% of the samples studied. In 60.4% cases, the combined expression of Oct4, Nanog and VEGF was noted, whereas in 52.8% cases, McM2, ABCG2 and VEGF were all positive. A statistically significant association was found between the prevalence of ABCG2, McM2 and VEGF (p = 0.000) and between Nanog, Oct4 and VEGF (p = 0.023). Conclusions: RT-‐PCR study on human retinoblastoma tissue revealed a high prevalence of ABCG2, McM2, Embryonic Stem Cell markers and VEGF. In addition, co-‐ existence of stem cell markers and VEGF was found in a high proportion of cases. Further studies to quantitatively assess these markers and to correlate their presence with clinical outcome and histopathologic features are recommended to determine their role in tumor behaviour and response to chemotherapy.

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Board #6 -‐ SUBRETINAL GENE THERAPY IN BARDET-‐BIEDL SYNDROME TYPE I MICE. ARLENE DRACK; SAJAG BHATTARAI; SEONGJIN SEO; DAN GRATIE; EDWIN STONE; VAL SHEFFIELD; ROBERT MULLINS, University of Iowa, Coralville, IA, USA Introduction: Bardet-‐Biedl Syndrome is a multisystem ciliopathy that includes an aggressive form of retinitis pigmentosa with childhood onset. Another childhood onset retinal disorder, Leber Congenital Amaurosis due to RPE65 gene mutations, shows improvement in both animal models and humans with the disease with subretinal gene replacement therapy. This study was designed to test the safety and efficacy of subretinal injection of AAV-‐WTBbs1 for the treatment of a mouse model of Bardet Biedl syndrome type 1 (BBS1). Materials & Methods: Bbs1M390R/M390R mice were generated as described previously. Constructs were made of the wild type Bbs1 gene in AAV2/5 vectors with a chicken Î²-‐actin promoter both with and without a FLAG tag. Subretinal injections were given through a scleral puncture under the temporal retina at P30 to P60 using AAV-‐WTBbs1 in the right eyes and sham injection in the left. One to 3 microliters were delivered including 1 part AAV-‐GFP and 9 parts AAV-‐WTBbs1 to enable visualization of the transduced area. Transgene expression was analyzed by immunohistochemistry and Western blotting following sucrose gradient ultracentrifugation. Retinal function was analyzed by electroretinogram, and structure was analyzed by optical coherence tomography (OCT). Histology was performed on selected animals at different time points. Results: Expression of FLAG-‐tagged Bbs1 was demonstrated in photoreceptor cells using antibody directed against the FLAG tag. Co-‐injection of AAV-‐GFP demonstrated transduction of 24-‐32% of the retina. Western blotting demonstrated BBS1 protein expression and reconstitution of the BBSome. ERG dark-‐adapted bright flash b-‐wave amplitudes were higher in AAV-‐Bbs1 injected eyes than in sham injected fellow eyes in more than 50% of 19 animals. Anti-‐rhodopsin staining demonstrated improved localization of rhodopsin in AAV-‐Bbs1 treated eyes. WT retinas injected with AAV-‐Bbs1 with or without a FLAG tag showed outer retinal degeneration on ERG, OCT and histology. Conclusions: In a knock-‐in model of BBS1, subretinal delivery of AAV-‐Bbs1 rescues BBSome formation and rhodopsin localization, and shows a trend toward improved ERG. However, whether this would have a clinically significant effect on vision is unknown due to concomitant overexpression toxicity. In WT mice with normal amounts of BBS1 protein, providing additional BBS1 protein damages the outer retina. In Bbs1 knock-‐in mice, which lack normal protein, there is only a small therapeutic effect. BBS is challenging to treat with gene therapy due to the stoichiometry of the BBSome protein complex and overexpression toxicity. Board #7 -‐ EXOME SEQUENCING IDENTIFIES RDH12 COMPOUND HETEROZYGOUS MUTATIONS IN A FAMILY WITH SEVERE RETINITIS PIGMENTOSA. JUAN CARLOS ZENTENO; OSCAR CHACON-‐CAMACHO; BEATRIZ BUENTELLO-‐VOLANTE; JONATHAN

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QUEVEDO-‐MARTÃ-‐NEZ; SERGUE JITSKII, Institute of Ophthalmology, Conde de Valenciana, Mexico City, Mexico Introduction: Retinitis pigmentosa (RP) is an inherited retinal dystrophy caused by a progressive and irreversible loss of photoreceptors. RP prevalence is approximately 1/4000 worldwide and it may be transmitted in all inheritance patterns. To date, 36 genes have been associated with autosomal recessive retinitis pigmentosa. Due to arRPâ?Ts phenotypic and genetic heterogeneity, its molecular diagnosis is highly complex and time-‐consuming. The aim of this work was to identify the causative mutations in a Mexican inbred family with arRP. Materials & Methods: A total of 4 affected siblings were clinically evaluated. Patients underwent full ophthalmologic examination including fundus examination, ERG, FAG, and OCT. Molecular analysis was performed in genomic DNA from affected subjects and a number of non-‐affected relatives. A genome wide linkage analysis was performed on DNA from 3 patients by means of an Affymetrix 250K single-‐nucleotide polymorphism microarray. Exome sequencing of one affected family member was done using the TrueSeq exome enrichment kit, captured using Nimblegen SeqCap EZ V2.0 probes kit, and paired-‐end sequenced on an Illumina Hiseq 2000 platform at Ambry Genetics (Aliso Viejo, CA, USA). Dideoxy sequencing was used for candidate gene variant confirmation. Results: All affected patients exhibited dense intraretinal pigment migration, severe retinal pigment epithelium atrophy, and arteriolar attenuation, with a severe atrophic pigmentary maculopathy. A large region of homozygosity was observed at chromosome 18q. The TUBB6 gene was sequenced as a candidate gene but no mutations were demonstrated. Exome sequencing analysis disclosed two pathogenic mutations in the RDH12 gene: c.295C>A (p.L99I) and c.446T>C (p.L149P). Sanger sequencing demonstrated segregation of both RDH12 mutations in all affected subjects and mutation heterozygous status in obligate carriers. Conclusions: While homozygosity mapping is an effective tool for identification of the underlying causative gene in inbred families, compound heterozygosity may occur even within the same consanguineous family. This works support that whole exome sequencing is a powerful and cost-‐effective tool for molecular diagnostics in families with inherited retinal dystrophies. Board #8 -‐ HOMOZYGOSITY MAPPING IDENTIFIES THE CLN3 GENE AS RESPONSIBLE FOR A RECESSIVE FORM OF RETINITIS PIGMENTOSA. BEATRIZ BUENTELLO VOLANTE; RAUL AYALA-‐RAMIREZ; JUAN C. ZENTENO, Institute of Ophthalmology â?oConde de Valencianaâ, Mexico City, Mexico Introduction: Retinitis pigmentosa (RP) is an inherited retinal disorder in which progressive loss of photoreceptors, primarily rods, leads to nyctalopia, peripheral visual field loss, and ultimately to blindness. The genetic basis and clinical phenotypes of RP are highly heterogeneous. Currently RP is known to be caused by mutations in over 50 genes and its inheritance is complex, including autosomal dominant (ad), autosomal recessive (ar), X-‐linked (xl), digenic, and even mitochondrial forms. Homozygosity mapping is a powerful approach for the identification of arRP causative genes. In this work, the identification of a homozygous CLN3 mutation in an arRP case is presented.

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Materials & Methods: The patient is a 10-‐year-‐old female without familial history of visual deficit and product of a consanguineous (uncle-‐nephew) marriage. Her health was unremarkable until the age of 7 years, when she was noted to have visual loss and after ophtalmological examination, a diagnosis of early onset RP was made. At examination, visual acuity was hand movements and fundoscopic examination disclosed mottled pigmentation in the macular area, few bone spicules in peripheral retina, mild attenuation of the retinal vessels and normal optic disc, in both eyes. No neurologic symptoms were evident and she was performing well at school. Genomic DNA was obtained from the patient and her parents and a genome-‐wide linkage scan using an Affymetrix 250K SNPs array was undertaken to identify regions of homozygosity. HomozygosityMapper and GeneDistiller softwares were used for the identification of candidate regions and genes, respectively. PCR amplification and Sanger sequencing were used for confirmation of possible pathogenic mutations. Results: Three chromosomal regions of maximal homozygosity were identified. The widest region (8.02 Mb) corresponded to chromosome 16, from base 25,911,212 to base 33,940,200, and includes the CLN3 gene that extends from base 28,488,600 to base 28,503,623. Mutations in CLN3 were screened by direct sequencing revealing a homozygous missense variant c.G565C:p.G189R, in patients DNA. The allele was predicted to be damaging based on our in silico analyses. Parental segregation test further support the pathogenicity of the variant. Conclusions: CLN3 is an endosomal/lysosomal transmembrane protein mutated in classical juvenile onset neuronal ceroid lipofuscinosis, commonly referred to as Battenâ?Ts disease, a fatal inherited neurodegenerative lysosomal storage disorder. Our results indicate that CLN3 is also involved in non-‐syndromic forms of retinitis pigmentosa. This case adds another example of the usefulness of homozygosity mapping for identification of causal arRP genes. Board #9 -‐ NOVEL AND KNOWN FRMD7 MUTATIONS IN BELGIAN PATIENTS WITH X-‐LINKED IDIOPATHIC INFANTILE NYSTAGMUS. BASAMAT ALMOALLEM; PATRICIA DELBEKE; JULIE DE ZAEYTIJD; BART P. LEROY; ELFRIDE DE BAERE, Ghent University, Ghent, Belgium Introduction: Idiopathic Infantile Nystagmus (IIN [MIM# 310700]) is characterized by infantile nystagmus and relatively good visual acuity. A family history of X-‐linked inheritance suggests FERM domain-‐containing 7 gene (FRMD7)-‐related infantile nystagmus (FIN), whereas IIN without a family history suggests non-‐FRMD7 IIN. Up to date 45 unique FRMD7 mutations have been reported in FIN patients. In this study, it was our aim to search for FRMD7 mutations in a Belgian cohort of 28 unrelated individuals with a clinical diagnosis of IIN. Materials & Methods: Detailed ophthalmological examinations were performed. Genomic DNA was extracted from the peripheral blood of the affected members. The entire coding region and splice junctions of the FRMD7 gene were investigated using polymerase chain reaction (PCR) followed by Sanger sequencing. The reference sequence used is NM_194277.2.

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Results: FRMD7 mutations were found in six unrelated index patients, five of which are unique. One of them is a known missense mutation c.70G>A (p.Gly24Arg), previously reported in Chinese pedigree with FIN. It is located in a highly conserved residue, up to C. elegans. The Grantham distance is 125. Polyphen, SIFT and MutationTaster predictions are suggestive for an effect on protein function. In the remaining five families four novel mutations were found: (i) c.660del (p.Asn221IlefsX11), found in a carrier mother and an affected male respectively, creates a frameshift in codon Asn221, resulting in a stop codon 10 amino acids downstream; (ii) c.2036del, found in an affected male, creates a frameshift from codon Leu679, resulting in a stop 7 positions downstream (p.Leu679ArgfsX8). Both deletion transcripts are likely subjected to nonsense mediated decay; (ii) missense mutation c.801C>A (p.Phe267Leu) was found in an affected female. It changes a strongly conserved residue (up to C. elegans). The Grantham distance is 22 (<60). Polyphen, SIFT and Mutation Taster predict an effect on protein function; (iv) one splice site mutation c.497+5G>A was found in an affected male. The effect of this donor splice site was not conclusive following splice site predictions. Segregation testing of these mutations is ongoing in order to further evaluate the clinical significance of these mutations. Haplotype analysis will be performed of mutation c.660del found in two independent families. Conclusions: We found FRMD7 mutations in 6/28 Belgian families with INN (21%). Our study provides additional evidence for FRMD7 mutations as a common cause of IIN and expands the mutational spectrum of FIN. Board #10 -‐ EXOME SEQUENCING IDENTIFIES A NOVEL RP1 MUTATION IN A BELGIAN FAMILY WITH AUTOSOMAL DOMINANT RETINITIS PIGMENTOSA. CAROLINE VAN CAUWENBERGH, GHENT, BELGIUM Board #11 -‐ VARIABLE PHENOTYPE IN ENHANCED S-‐CONE SYNDROME: BARBARA JANSSENS; JULIE DE ZAEYTIJD; FRAUKE COPPIETERS; ELFRIDE DE BAERE; BART PETER LEROY, Department of Ophthalmology, Ghent University Hospital, Ghent, Belgium Introduction: To study the phenotypes of 5 patients with enhanced S-‐cone syndrome (ESCS) and known NR2E3 mutations, and evaluate whether genotype-‐phenotype correlations exist. Materials & Methods: Five patients with ESCS underwent an extensive ophthalmological examination, including blue light autofluorescence imaging (BAF), optical coherence tomography (OCT) and electroretinography (ERG). Bidirectional sequencing of the entire coding region and the intron-‐exon boundaries of the NR2E3 gene was performed. Results: All patients were male. The age at presentation varied between 25 and 37 years. Best-‐corrected visual acuity (BCVA) ranged from counting fingers (CF) to 10/10. One patient was followed up for 23 years during which his BCVA dropped to CF. Four patients showed large, nummular clumps of pigmentation around the vascular arcades. One patient showed central macular atrophy, two had a normal macula, whilst one patient had a mild and another severe macular schisis on OCT. All patients had a

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characteristic full-‐field ERG with absence of rod responses and a large but delayed dark-‐adapted response to an intense flash, which was near identical to that of the cone-‐related photopic response to a transient intense flash. BAF showed alternating hypo-‐ and hyperautofluorescent flecks around the vascular arcades, whereas the central macula was either normal or showed hyperautofluorescent spots or alternating hypo-‐ and hyperautofluorescent concentric rings. Three patients were compound heterozygous for the following respective mutations: c.119-‐2A>C in intron 1 and c.290G>A in exon 3; c.309C>A in exon 3 and c.639_640insT in exon 5; c.208T>C in exon 2 and c. 119-‐2 A>C. Two patients were homozygous for the following respective mutations: c.1037A>G in exon 7 and c.119-‐2A>C in intron 1. The clinical phenotype as measured with BCVA, OCT, BAF and fundoscopy was most severe in the patient who was homozygous for the splice site mutation c.119-‐2A>C in intron 1, and least severe in the patient who was compound heterozygous for c.208T>C in exon 2 and c. 119-‐2 A>C, with that of the others in between. However, this was not reflected in the ERG results. Conclusions: ESCS is a progressive retinal dystrophy with a variably severe phenotype, characterized by nummular areas of hyperpigmentation around the vascular arcades, macular schisis and a typical ERG pattern. We were unable to establish unequivocal genotype-‐phenotype correlations in our small cohort. Board #12 -‐ OCULAR PHENOTYPES OF JALILI SYNDROME. SOFIE GEENS; FRAUKE COPPIETERS; SOFIE WALRAEDT; ELFRIEDE DE BAERE; BART PETER LEROY, University Hospital Ghent, Ghent, Belgium Introduction: To describe the clinical phenotype in five patients from two families with Jalili syndrome caused by mutations in CNNM4. Materials & Methods: Five patients with Jalili syndrome underwent an extensive ophthalmological examination, including blue light autofluorescence imaging (BAF), optical coherence tomography (OCT) and full-‐field flash electroretinography (ERG). Bidirectional sequencing of the entire coding region and the intron-‐exon boundaries of the CNNM4 gene was performed. Results: A total of 3 female and 2 male patients were examined. Ages at visits ranged between 6 months and 27 years. Four patients were from one family, the other an unrelated simplex case. The longest follow up period was 26 years. All patients initially presented with photophobia and congenital nystagmus. Best-‐corrected visual acuities ranged from 8/100 to 1.5/10 and slowly declined over time. All patients were hypermetropic. Colour vision showed acquired achromatopsia. Goldmann visual fields were full in the early stages, but showed slowly progressive peripheral loss during evolution. Fundoscopy, BAF and OCT showed outer retinal atrophy in the central macula. As the condition progressed, outer retinal atrophy became more evident, first at the macula and then more widespread to gradually include the periphery. The youngest patient was first seen at the age of 6 months, with central macular abnormalities already apparent. The solitary patient from family 2 showed remarkable sparing of the superior retina separated from the central area of bull's eye atrophy by a discrete demarcation line. ERGs were performed in four of the five patients. Before the

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age of 10 years, ERGs were compatible with cone-‐rod dystrophy, whereas at a later age these were extinguished. All patient had considerable dentinogenesis imperfecta of both deciduous and permanent teeth, with severe quality loss and discolouration as telltale signs. The four patients from family 1 were homozygous for the c.189del mutation in CNNM4. All were born from consanguineous marriages. The simplex patient from family 2 was homozygous for the missense mutation c.1852G>C. No parental consanguinity was known. Conclusions: Early-‐onset cone-‐rod dystrophy in combination with dental abnormalities should prompt a diagnosis of Jalili syndrome. Nystagmus in the first months of life, with photophobia, central macular atrophy and low quality dentition, are typical features. Central vision is low from birth, but evolution of the retinal dystrophy is moderately slow, with remaining basic visual function beyond the age of 25 years. Biallelic mutations in the CNMM4 gene are the molecular cause of this condition. Board #13 -‐ OCULAR DEVELOPMENT AND AXIAL LENGTH IN THE BESTROPHINOPATHIES. JULIE DE ZAEYTIJD, TINE SABBE, ELFRIDE DE BAERE, BART LEROY, Ghent University Hospital, Ghent, Belgium Introduction: To describe the relationship between the retinal pigment epithelium (RPE) and ocular development in the bestrophinopathies. The bestrophinopathies are a group of three diseases in which the pathogenic defect is located at the level of the RPE: Best vitelliform macular dystrophy (BVMD), autosomal dominant vitreoretinochoroidopathy (ADVIRC) and autosomal recessive bestrophinopathy (ARB). Materials & Methods: Thirty patients with BVMD, 15 patients with ADVIRC and 8 patients with ARB, with proven BEST1 mutations, underwent a detailed ophthalmological examination, including measurement of axial length and corneal diameter. In addition, psychophysical and electrophysiological testing (ISCEV-‐standard ERG, PERG and EOG) was performed in the majority of cases. Results: With a mean value of 21.8mm, the axial length is significantly shorter in both BVMD and ARB, than the normal mean value of 23.6mm. In ADVIRC patients, ocular length showed a mean value of 23.4mm, a difference that was not statistically significant. In ADVIRC, the corneal diameter is reduced with a mean value of 9.7mm. In BVMD and ARB with a mean value of respectively 10.7mm and 11.2mm, microcornea is less pronounced. Visual field defects were limited to (peri)central defects in addition to enlarged blind spots in all ADVIRC patients, and (peri)central sensitivity loss in ARB patients. Full-‐field flash ERGs were normal in 10/10 eyes with BVMD, abnormal in 24/30 eyes in ADVIRC and 12/16 eyes in ARB. A Lp/Dt-‐ratio < 150% on EOG was seen in 48/60 eyes with BVMD, and all patients with ADVIRC and ARB. Conclusions: These results show that ocular development is abnormal in the bestrophinopathies and thus suggest that either the presence of an abnormal bestrophin protein, or the complete absence of it in the RPE, influences ocular development. The normal axial length in ADVIRC is rather unexpected since the disease has been associated with ocular developmental problems. In BVMD and ARB, the axial

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length is significantly shortened. Microcornea is seen in all three conditions, but is more pronounced in ADVIRC. Board #14 -‐ UNILATERAL X-‐LINKED RETINOSCHISIS. MICHAEL RITZ; YAUMARA PERDOMO; GEORGES CAPUTO; CLAUDE SPEEG-‐SHATZ; HELENE DOLLFUS, Hopital Civil, Strasbourg, France Introduction: X-‐linked retinoschisis (XLRS) is a genetic ocular disease characterized by microcystic changes of the macular region, peripheral schisis and vitreous veils. These lesions are responsible for impaired vision and are usually bilateral and symmetrical. We report on an unusual case of unilateral XLRS. Materials & Methods: A 7-‐month-‐old boy was referred to his ophtalmologist because of strabismus of the right eye. The examination during general anaesthetic showed a vast retinal cyst in the lower region of the right eye with a pigmented line in the macular region. Slit lamp biomicroscopy of both eyes was unremarkable, and the fundus examination of the left eye was normal.The patient was diagnosed with XLRS, in spite of the apparent unilaterality. Results: The molecular analysis of the RS1 gene identified the mutation c.589C>T (p.Arg197Cys) in exon 6. This mutation was previously described by the Retinoschisis Consortium and is considered as probably deleterious by the prediction software. The mutation is carried by the mother. Conclusions: Taking into account the unusual unilateral clinical presentation, the heterogeneous natural history of X-‐linked schisis in the litterature, and the demand of the parents for future prenatal diagnosis or pre implantatory diagnosis, our team experimented major ethical questioning about this case and seek by way of this poster for other teams with similar cases. Board #15 -‐ RETINAL HISTOPATHOLOGY IN EYES FROM PATIENTS WITH AUTOSOMAL RECESSIVE RETINITIS PIGMENTOSA CAUSED BY EYS MUTATIONS. PAUER GJ, BONILHA VL, RAYBORN ME, BELL BA, MARINO, MJ, BEIGHT CD, CHIANG J, TRABOULSI EI, HAGSTROM SA, HOLLYFIELD JG, Cleveland, USA Purpose: To evaluate the histopathology in donor eyes from patients with autosomal recessive retinitis pigmentosa (arRP) caused by mutations in the EYS gene. Methods: Eyes were obtained from a 72 year-‐old female (donor 1), a 91 year-‐old female (donor 2), and her 97 year-‐old sister (donor 3). Eyes were fixed in 4% paraformaldehyde and 0.5% glutaraldehyde in PBS within 6 to 11 hours postmortem. Globes were evaluated with macroscopic, SLO and OCT imaging. Macular and peripheral regions were processed for electron microscopy and immunocytochemistry. Three age-‐matched normal eyes and an eye donated by donor 1’s asymptomatic mother (donor 4) were used as controls. DNA was obtained from blood samples of the donors for mutation analysis. Results: DNA analysis of the affected brother of donor 1 revealed the EYS gene mutations, IVS11+1G>A and Q874X. DNA analysis of donors 2 and 3 led to the identification of a heterozygous 7 base pair deletion, L1451fx, in exon 26 of the EYS

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gene. The histopathological findings of the retina of donor 1 and her asymptomatic mother (donor 4) were reported previously and are here compared to the findings in the retinas of donors 2 and 3. SLO of donor 3 showed hypofluorescence in one contiguous region involving the optic disc and macular region as opposed to the individually demarcated and isolated region seen in donors 1 and 2. Histology of all three affected donors revealed a highly degenerated retina with little evidence of stratified nuclear layers in all peripheral areas studied. In contrast, the macular area displayed a prominent inner nuclear layer. Donors 2 and 3 revealed patchy areas of RPE in the macular whereas the RPE was thin in the macula and absent in the far periphery of donor 1. Rhodopsin-‐labeled rods were absent except in the far periphery of dono0r 1. However, rods were still present in the periphery of donors 2 and 3. Of interest, some rods were observed in the choroid of these eyes. In the macula, donor 2 displayed no rods while donor 3 displayed several disorganized rods. Cone arrestin-‐labeled cells were present in the macula, but were mostly absent from the periphery of donor 1. Patches of cones were observed in the perimacular region of both donors 2 and 3. Conclusions: Advanced retinal degenerative changes with near-‐total absence of rods and preservation of macular cones characterize the retinal histopathology of arRP patients due to EYS gene mutations. Board #16 -‐ GENETIC COUNSELLING REMAINS CHALLENGING IN LEBER’S OPTIC NEUROPATHY. VALERIE PELLETIER; PHILIPPE GOSSET; MARGUERITE MIGUET; PERDOMO YAUMARA; DOLLFUS HELENE, Hopital Civil, Strasbourg, France Introduction: Leber’s hereditary optic neuropathy (LHON) is usually characterised by initial unilateral subacute central visual loss with rapid bilateralisation leading to a severe visual handicap. This optic neuropathy is exclusively transmitted by the women because of mitochondrial inheritance. More than 18 homoplasmic mutations are identified today. The age of onset varies from infancy to late adulthood. The neuropathy is usually isolated but can be rarely associated to cardiac conduction defects or neurologic abnormalities. The risk of developing this condition differs between genders, ranging from 30% to 83% in men to 5% to 32% in women. Materials & Methods: The homoplasmic concept, the risk of an abrupt severe visual impairment (which is four to five times higher in men than women), the guilty feeling of the mothers who carry a LHON mutation and the feeling of a “sword of Damocles” over their children’s head are major issues for genetic counseling that remains a challenge for women who wish to have children. Results: We report four clinical situations that illustrate the complexity of genetic counseling for LHON: in a context of a prenatal project or during a pregnancy, in the context where another genetic affection coexists. In all cases, questions about prenatal diagnosis (PND) and preimplantation genetic diagnosis (PGD) were addressed. Conclusions: What is the place for sex determination by PGD? What about PND? We will detail the outcomes of each situation that learns us about the deep impact of LHON in the families.

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Board #17 -‐ CRANIOFRONTONASAL SYNDROME IN A MALE WITH SAGITTAL CRANIOSYNOSTOSIS. JACQUELINE HOOVER; HANNAH L. SCANGA; KEN K. NISCHAL, Children's Hospital of Pittsburgh of UPMC, Pittsburgh, PA, USA Introduction: Craniofrontonasal syndrome (CFND) is a rare genetic developmental disorder with an unusual pattern of X-‐linked inheritance showing greater severity in heterozygous females than in hemizygous males (1). Here we report a 7-‐year-‐old male with midface hypoplasia, prognathia, and sagittal synostosis with a more mildly affected mother. Both have molecularly confirmed CFND. Materials & Methods: Retrospective review of clinical details and mutation data of a trio referred for ophthalmology evaluation. Results: A 7-‐year-‐old male, previously followed by a craniofacial department and a geneticist, presented for ophthalmology evaluation. His initial symptoms included headaches and abnormal head shape. CT scan revealed fusion of the sagittal suture, mild left occipital plagiocephaly, and prognathia with crowding of the teeth in the lower mandible and maxilla. His intraoral exam revealed an intact palate with Class III malocclusion. His dysmorphology examination was significant for an inner canthal and interpupillary distance greater than the 97th percentile, ptosis with upslanting palpebral fissures, large ears (>97th percentile), and a broad nasal root and bridge. Previous genetic testing included normal male chromosome analysis and oligoarray. No mutations were detected on sequencing of select exons of FGFR2. Ophthalmology examination was negative for papilledema, but noted exocyclotorsion of the fundus, and unusual movements with gaze. A family history was obtained, where his mother was noted to have hypertelorism, broad nasal tip, grooved fingernails, and umbilical hernia. Concern for X-‐linked inheritance, specifically CFND, was raised. Genetic testing was subsequently offered for this child. Sequencing of exons one through five of the EFNB1 gene revealed a novel C to T mutation at nucleotide 466 (c.466C>T). Bioinformatics models (SIFT and Mutation Taster) predicts c.466C>T to be likely pathogenic. Family testing was pursued and the patientâ?Ts mother was confirmed to carry the c.466C>T mutation, while his unaffected brother did not carry the mutation. Conclusions: CFND is an inherited disorder characterized by short stature, hypertelorism, bifid and broad nasal tip, coronal craniosynostosis in females, syndactyly, and grooved nails with longitudinal splitting. Associated with mutations in one of five exons of ephrin B1 (EFNB1) at chromosome Xq13.1, 42% of mutations are missense changes in exons 2 or 3 that encode the extracellular ephrin domain (1). Typically, expression is more severe in females and this has been demonstrated in female heterozygous mice, where they express the skeletal phenotype while hemizygous males and homozygous females do not (2, 3). A review of literature did not reveal sagittal synostosis in molecularly confirmed affected males, suggesting that this case represents a previously unrecognized male phenotype of CFND. This is significant when providing genetic counseling to mothers with confirmed EFNB1 mutations. Ultimately, the familial case presented here highlights the importance of considering this potential diagnosis in males with craniosynostosis, exocyclotorsion of the fundus, and unusual eye movements.

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References 1.. Zafeiriou DI, Pavlidou EL, Vargiami E. Diverse Clinical and Genetic Aspects of Craniofrontonasal Syndrome. Ped Neur 2011 Feb; 44(2):83-‐7. 2. Davy A, Aubin J, Soriano P. Ephrin-‐B1 forward and reverse signaling are required during mouse development. Genes Dev 2004; 18: 572-‐583. 3. Wieland I et al. Mutations of the Ephrin-‐B1 Gene Cause Craniofrontonasal Syndrome. Am. J. Hum. Genet. 2004; 74:1209-‐1215.

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