Giessen 2019 – Day 1

Giessen 2019 – Delegates explore Marburg

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Session 1 – Performing And Communicating Molecular Diagnostics

Welcome address by the President of ISGEDR and the Speaker of the Section DOG-Genetics

Dear colleagues, dear friends, dear supporters,

For the second time since its inauguration in 2011, the section DOG-Genetics has joined with an international society to present you a multidisciplinary meeting on genetic eye disorders. I am delighted and curious by the opportunities emerging from the cooperation with the International Society for Genetic Eye Diseases and Retinoblastoma (ISGEDR) in organizing an international meeting. A large number of internationally highly renowned scientists have followed our invitation to give an overview on their most recent cutting edge research. In 34 invited lectures, the state of the art in the identification of genetic causes of inherited retinal denerations (IRD), the development of biomarkers to characterize the progression of IRDs, and the current developments in retinoblastoma diagnostics and management will be presented. The discussion of treatment options for both types of eye disorders will summit in an expert panel on the first approved and commercially available gene therapy for one form of IRD.

The highlights of the program are three prestigious lectures established by ISGEDR.
The Franceschetti lecture is given by the outgoing director of the US National Eye Institute (NEI) newly appointed director of the Center for Ocular Regenerative Therapy at UC Davis, Prof. Paul Sieving on “Clinical features and molecular basis of X-linked retinoschisis: From mechanism to therapy”. Along with the lecture, Prof. Sieving will be honored with the Franceschetti medal for his extraordinary achievements in research on IRDs and the development of treatment options.

The Ellsworth lecture is dedicated to retinoblastoma research and will be presented this year by Dr. Laurence Desjardins, a director of the institute Curie in Paris and on the topic of “Retinoblastoma around the world in 2019”.

Finally, Í am very delighted and extremely honored to have been selected to present this year´s François lecture. I will focus on “Biomarkers in IRDs: scientifically valid – clinically relevant” as an extremely important prerequisite for all treatment approaches.

The scientific program further encompasses 32 free paper presentations and 42 free poster presentations from basic and clinician scientists, bringing together researchers from 21 countries from all over the world to share their knowledge. The mixture of participants ensures a vital communication and spreading of knowledge throughout all countries involved in research for inherited eye disorders.

I am grateful to all contributors whose efforts helped in establishing the programme starting with the scientific committee (Elias Traboulsi, Francis Munier, Brenda Gallie, Knut Stieger, Markus Preising, Walter Lisch, and Dietmar Lohmann) and the local organizational team. In this respect, my very special thanks go to Markus Preising who has really accomplished most of the organizational work to make the meeting happen as it is.

Major contributions from the German Research Foundation (DFG) and the German Ophthalmological Society (DOG) helped us to gather together experts from all over the world and to honor six young scientists with travel grants. This year, we were able to double the number of travel grants (usually 3 issued by ISGEDR) through the support of the DOG.

Finally, without the generous support of our commercial partners we would not have been able to establish the local setting in the pleasant way as it is. Therefore, I recommend visiting the industrial exhibition at the ground floor and express my gratitude to all our sponsors.

I am extremely delighted by the large number of participants who followed our invitation and wish all of you a pleasant stay, exciting discussions, beneficial encounters for your scientific success and stimulating exchanges with old and new friends.

Birgit Lorenz
President of ISGEDR
Speaker of the DOG Section Genetics

NGS: Diagnostic opportunities and challenges of gene panel, whole-exome and whole-genome sequencing

Purpose: Next-generation sequencing (NGS) has transformed research and diagnostics of hereditary disorders. This presentation will show that this is particularly true for eye diseases because many of them have a monogenic basis.

Methods: The opportunities and challenges of current diagnostic applications of NGS – gene panel, whole-exome (WES) and, occasionally, whole-genome sequencing (WGS) – are discussed based on examples from the literature and own experience, mostly from the genetically most heterogenic group, the retinal dystrophies.

Results: The diagnostic application of NGS results in a high diagnostic yield. While the causative mutation is easy to pinpoint in many patients, some cases are challenging regarding potential pitfalls and misleading “hits”. The discovery of new genotype-phenotype correlations and, depending on the NGS approach, of new disease genes is demonstrated. Some genes are candidates for therapeutic approaches that may not be restricted to classical gene therapy.

Conclusions/Significance: While generating a patient’s genetic data is no longer the analytical bottleneck, data interpretation requires profound knowledge of the genetic and phenotypic characteristics and close cooperation between geneticists and ophthalmologists in order to draw the correct conclusion from the challenging complexity of genetic data. The diagnostic application of even genome-wide NGS approaches such as whole-exome and whole-genome sequencing has now blurred the borders between diagnostics and research. This offers new opportunities, including the discovery of new disease genes. The information on the causative gene enables personalized medical follow-up that may, in case of syndromic entities, require the involvement of other clinicians than ophthalmologists. As the first gene therapies are being applied, the genetic analysis has gained a potential therapeutic dimension.

Conflict of interest disclosure: none

Predictive testing in glaucoma

David Mackey
University Of Western Australia, Subiaco, Australia

Purpose: To present the current state of predictive DNA testing for glaucoma.
Methods: Data from the UK biobank UKBB and International Glaucoma Genetics Consortium IGGC suggest that multiple genetic risk alleles contribute to glaucoma. Over the last two decades we have been able to provide predictive DNA testing for a small number of families with Mendelian fors of glaucoma such as MYOC, OPTN and MCCRP3.

Results: SNPs near over 100 genes have now been associated with genetic risk for Primary Open Angle Glaucoma.

Conclusions: Although we could provide genetic risk scores in a similar way to glaucoma clinical risk scores, we need to test the predictive power of genetic risk scores in other populations, involving both people with a family history of glaucoma and the general population.

Conflict of interest disclosure: none

We need more genetic counselors!

Jenina Capasso
Wills Eye Hospital, Philadelphia, PA, USA

As genetics integrates into nearly every subspecialty of medicine, it becomes increasingly important to have genetic counselors in clinical practice. Abacan et al., reported on the global state of the profession in 2018, with only a few countries meeting the work force recommendation of 6-12 counselors/million population. The number of genetic counselors in North America is estimated around 5,000 and those who work in ocular genetics or with some overlap is approximately 50. At Wills Eye Hospital, we have trained several fellows who return to practice in countries with little to no access to genetic counselors. This talk aims to describe the current under availability of genetic counselors in ocular genetics subspecialty and propose a model for training more counselors globally.

Reference: Abacan M. et al. The Global State of the Genetic Counseling Profession. Eur J Hum Genet. 2019 Feb;27(2), 183-197.

Conflict of interest disclosure: none

Interrogation of the 100,000 genomes project ophthalmic disease cohort reveals novel genes, new associations and previously undetectable mutations

Gavin Arno1
Ba-Abbad, Rola1; Jurkute, Neringa1; Mahroo, Omar1, 2; Moosajee, Mariya1; Yu Wai Man, Patrick1, 3; Michaelides, Michel1; Webster, Andrew R.1
1. UCL Institute Of Ophthalmology And Moorfields Eye Hospital, London, UK
2. Department Of Ophthalmology, St. Thomas’ Hosp, London, UK
3. Mitochondrial Biology Unit, MRC And Cambridge Centre For Brain Repair , London, UK

Purpose: To characterise pathogenic variants in whole genome sequencing (WGS) data from a cohort of patients with a broad spectrum of ophthalmic disorders.

Methods: Patients and families were recruited from the inherited eye disease clinics at Moorfields Eye Hospital, London as part of the UK 100,000 genomes study. A cohort of 420 probands with ophthalmic disease underwent clinical variant interpretation according to the American College of Medical Genetics guidelines and discussion at a multidisciplinary meeting following WGS and automated variant prioritisation in a curated virtual gene panel ( Unsolved cases were selected for research including non-coding/structural variant analysis in the gene panel, new gene/disease associations and novel gene investigation. Potential pathogenic structural and non-coding variants were selected for functional analysis by applying an integrated analysis pipeline incorporating deep phenotyping and variant filtering and interpretation tools in patients unsolved following coding variant analysis. Variant effects were confirmed where possible using molecular biology techniques.

Results: To date, our analysis pipeline has identified likely pathogenic genotypes in 253/420 (60%) probands. These include more than 30 patients habouring variants in unexpected or novel genes or non-coding/structural variants across the virtual gene panel, including: 1. splice region and deep intronic single nucleotide variants that give rise to altered splicing. 2. upstream gene regulatory region variants that alter the level of gene transcription through changes in transcription factor binding sites. 3. deletion/duplication affecting at least one coding exon across the gene panel.

Conclusions: Implementation of WGS in a clinical genomic pipeline enables detection and interpretation of potential pathogenic variants across the entire genomic footprint of a diagnostic gene panel. Unsolved cases underwent expanded variant discovery analysis yielding a significant number of additional findings. We report newly identified variants otherwise missed by exon-focused diagnostic strategies that account for a significant proportion of missing heritability in IRD. In silicoand functional investigation confirms the pathogenicity of these variants and should be integrated in future clinical diagnostic pipelines incorporating WGS screening.

Conflict of interest disclosure: none

The value of CNV analysis for inherited retinal diseases

Meghan Debenedictis
Traboulsi, Elias
Cole Eye, Cleveland Clinic, Cleveland, OH, USA

Multiple genetic testing labs offer large retinal dystrophy NGS panels and some include CNV analysis. Utilizing a panel approach that includes CNV analysis has shown to be valuable in identifying etiology of disease. The following cases highlight the importance of genetic testing, including CNV analysis, for patients with retinal dystrophies. The first case is an 8 year male with an onset of vision loss at age 4. He had no systemic health problems. The family history was negative for vision loss or genetic disease. He was diagnosed with Leber congenital amaurosis by an ophthalmologist. A 31 gene LCA NGS panel was negative. Subsequent testing via a 280 gene retinal dystrophy panel with CNV analysis identified him to have a heterozygous c.1056+3A>C pathogenic variant in the CLN3 gene. Additionally, CNV analysis identified him to have a 0.3 kb deletion in CLN3. This test result is consistent with a diagnosis of Juvenile neuronal ceroid lipofuscinosis. He was referred to a Batten disease center of excellence for management. The second case is a 41 yr female who became symptomatic at age 31 with nyctalopia and decreased peripheral vision. Ophthalmic exam was consistent with RP. Medical history was unremarkable. She had undergone genetic testing for a panel of 13 recessive RP genes in 2012 which was negative. NGS of a larger 262 gene retinal dystrophy panel was performed in January, 2016, which identified a homozygous c.783G>A pathogenic variant in the CDHR1 gene, consistent with recessive RP. Two years later, results from prior participation in a research study (308 gene panel with CNV analysis) became available which identified her to be hemizygous for the CDHR1 variant. She also was found to have a 7.38 MB deletion on chromosome 10, which included the entire CDHR1 gene, in addition to the PTEN gene, among 10 others. She had a maternal history of uterine cancer and thyroid problems. She and her mother were referred to PTEN clinic for management.These are two of many cases where genetic testing profoundly impacts patient care and illustrate the medical necessity of pursuing a comprehensive test.

Conflict of interest disclosure: none

Modeling gene constraint in disease populations for clinical molecular diagnostics

Robert Hufnagel
Bryan, John; Woo, Geena; Guan, Bin; Mcgaughey, David
National Eye Institute, Bethesda, MD, USA

Purpose: Clinical molecular genetic testing interpretation depends primarily on individual variant-level information and healthy population frequency data. Often, novel variants lacking additional information needed to assign likely pathogenic or benign status are designated as variants of uncertain significance. To address this need, we generated and applied gene-level and disease-population allele frequency data to current genetic testing interpretation criteria.

Methods: Whole exome and genome datasets from gnomAD and publicly available genetic variant datasets of patients with retinal degenerations (RD) were downloaded and processed to generate individual variant and gene-level allele frequencies for subsequent visualization and analysis. Statistical enrichment was determined by Fischer’s exact test of independence with Bonferroni correction.

Results: Gene models comparing RD-population to general-population (gnomAD) allele frequency data permitted statistical analysis of variants enriched in RD. Individual gene models for autosomal dominant (PRPH2), autosomal recessive (ABCA4), and X-linked recessive (RS1) retinopathies exhibited strong correlation with manually curated variant interpretation. Genome-wide models further demonstrated independent enrichment of known disease genes in a large RD cohort. Next, gene constraint analysis of the general-population dataset was used to calculate total rare variation frequencies for missense, truncating, and synonymous variation for every gene below population frequencies typically used for clinical variant interpretation. Notably, genes frequently detected with novel variants in next-generation sequencing data for RD were among those with the highest rare gene-level variation frequencies.

Conclusions/Significance: Gene-level variant information and RD-population frequency data add confidence to genetic testing interpretation of known and novel variation by providing information beyond general population allele frequencies. By comparing gene constraint models with expected single-gene contributions to disease prevalence, we can better estimate the likelihood of pathogenicity for novel variants detected in patients with RD.

Conflict of interest disclosure: none

DNA testing in a series of 944 patients with inherited retinal dystrophies from a single German reference center

Ulrich Kellner1
Stöhr, Heidi2; Kellner, Simone1; Weinitz, Silke1; Farmand, Ghazaleh1; Lindau, Birgit1; Weber, Bernhard H.F.2
1. Rare Retinal Disease Center, Augenzentrum Siegburg, MVZ ADTC Siegburg GmbH, Siegburg, Germany
2. Institute For Human Genetics, University Regensburg, Regensburg, Germany

Purpose: To report the findings of DNA testing in a series of 944 patients with inherited retinal dystrophies (IRD) from a single German reference center.

Methods: Following ophthalmological diagnosis of inherited diseases based on clinical findings, retinal imaging and/or electrophysiological examinations, patients underwent molecular genetic evaluation based on the clinical diagnosis with direct Sanger sequencing or custom designed gene panel analysis. The number of genes tested per patient ranged from one to 124.

Results: In 944 patients (813 unrelated families) disease-causing mutations (1 mutation in adIRD or xIRD and 2 mutations in arIRD) were identified in 422 subjects (44.7 %; 357 families (43.9 %). Disease causing mutations were identified in 58 different genes, most frequently in ABCA4 (24.9 % of families), BEST1 (11.8 %), PRPH2 (9.2 %), RS1 (9.0 %), RPGR (7.0 %), USH2A (5,6 %), RHO (4.8 %), CHM (2.8 %), EYS (1.7 %), and PRPF31 (1.4 %). Together, mutations in these 10 genes were associated with 76.8 % of all solved cases. In addition, 44 patients with macular dystrophy or cone-rod dystrophy (4.7 %) were found to be monoallelic carriers of a disease-causing ABCA4 mutation and a further 31 patients were monoallelic carriers of one disease-causing mutation in other recessive genes.

Conclusion: Comprehensive DNA testing reveals a high number of cases solved due to gene mutations in known retinal disease genes. Delineation of the impact of these mutations on the clinical course of the disease can only be achieved in a large cohort with clinical longtime follow-up as the one presented in this work.

Conflict of interest disclosure: none

Enhancing diagnostic performance in inherited retinal diseases through advances in high resolution copy number detection and RPGR ORF15 sequencing

Kirsty Wells1
Kämpjärvi, Kati1; Guidugli, Lucia1; Sistonen, Johanna1; Känsäkoski, Johanna1; Sarantaus, Laura1; Västinsalo, Hanna1; Mehine, Miika1; Valori, Miko1; Sankila, Eeva-Marja2; Schleit, Jennifer1; Saarinen, Inka1; Muona, Mikko1; Myllykangas, Samuel1; Koskenvuo, Juha1; Tuupanen, Sari1; Alastalo, Tero-Pekka1
1. Blueprint Genetics, Helsinki, Finland
2. Helsinki University Eye Hospital, Helsinki, Finland

Purpose: Inherited retinal dystrophies (IRDs) are clinically and genetically diverse disorders. Therefore, a comprehensive genetic testing strategy is essential to maximise diagnostic yield and select targeted therapies. However, due to technical limitations, genetic tests for IRD commonly do not capture the full spectrum of causative genetic variation. We aimed to develop a comprehensive next generation sequencing-based (NGS) RD panel, able to detect not only coding sequence alterations, but also copy number variants (CNVs), known pathogenic deep intronic variants, and variants in the difficult to sequence RPGR ORF15 hotspot. We then aimed to assess diagnostic yield and mutational spectrum in large RD cohorts, with emphasis on the prevalence and characteristics of CNVs and diagnostic RPGR ORF15 variants.

Methods: Diagnostic yield and mutational spectrum of sequence variants were evaluated in a cohort of 1587 RD patients, and tested using an NGS panel covering 266 IRD-associated genes. The rates and characteristics of CNVs were evaluated in a cohort of 2754 patients. Sequencing was performed by targeted OS-Seq using the Illumina NextSeq500 platform, or the IDT xGEN Exome Research Panel using the Illumina NovaSeq platform. CNVs were detected by CNVkit and an in-house developed deletion caller.

Results: The 266-gene RD panel yielded a diagnosis in 58% of cases. Diagnoses were made in 111 genes in total, the most frequently implicated genes being ABCA4, USH2A and RPGR. A molecular diagnosis in RPGR was identified in 5.7% of patients; 31% of RPGR diagnoses involved variants in the difficult-to-sequence central region of ORF15. In 4.6% of patients, a CNV matching the phenotype was reported; the majority (88%) being deletions. Of these, 49% were partial gene deletions, 25% one exon deletions, 12.5% whole gene deletions and 1.6% partial exon deletions. CNVs were identified in 47 genes, and USH2A and PRPF31 were enriched in CNVs compared to other genes.

Conclusions/Significance: Genetic testing of a large cohort of patients with IRD has revealed an important diagnostic contribution by CNVs and RPGR ORF15 variants. The broad mutation spectrum in our cohort demonstrates the importance of a comprehensive genetic testing approach in RD diagnostics, to optimize diagnostic yield and clinical care.

Conflict of interest disclosure: The following conflict(s) of interest must be disclosed:
Kämpjärvi, Kati; Guidugli, Lucia; Sistonen, Johanna; Känsäkoski, Johanna; Sarantaus, Laura; Västinsalo, Hanna; Mehine, Miika; Valori, Miko; Schleit, Jennifer; Saarinen, Inka; Muona, Mikko; Myllykangas, Samuel; Koskenvuo, Juha; Tuupanen, Sari; Alastalo, Te are employees of Blueprint Genetics, Helsinki, Finland

Session 2 – Clinical Studies in Gene Therapy I

Safety & efficacy of antisense oligonucleotide therapy (QR-110) in LCA10 patients with the c2991+1655A>G allele in CEP290

Bart P Leroy1
Cideciyan, Artur V2; Jacobson, Samuel G2; Ho, Alan C.3; Garafalo, Alexandra2; Roman, Alejandro J2; Schwartz, Mike4; Biasutto, Patricia4; De Wit, Wilma4; Cheetham, Michael E5; Adamson, Peter S4; Rodman, David4; De Zaeytijd, Julie1; Van Cauwenbergh, Caroline1; Drack, Arlene V.6; Russell, Stephen R.6
1. Dept Of Ophthalmology, Ghent University Hospital, Ghent, Belgium
2. Scheie Eye Institute, Univ Of Pennsylvania, Philadelphia, PA, USA
3. Wills Eye Hospital, Philadelphia, PA, USA
4. Proqr Therapeutics, Leiden, Netherlands
5. Institute Of Ophthalmology, Univ College Of London, London, United Kingdom
6. Dept Of Ophthalmology, Iowa University Hospitals, Iowa City, IA, USA

Purpose: to assess safety and efficacy of the antisense oligonucleotide QR-110 in LCA10 patients with the c.2991+1655A>G allele in the CEP290 gene.

Methods: in a 3-center open-label clinical trial (NCT03140969) subjects received intravitreal injections of QR-110 in the worse seeing eye. Systemic and ocular safety were evaluated using standard methods. Efficacy was evaluated with best-corrected visual acuity (BCVA), oculomotor control and instability (OCI), two-colour full-field sensitivity testing (FST) as well as mobility testing.

Results: 10 patients received between 1 and 4 injections and were followed for up to 9 months. At 3 months after the first injection, treated eyes showed significant improvement compared to baseline by an average of 0,67 log (7 lines), 0,62 log in red FST and 0,81 log in blue FST. At 3 months after the second injection, BCVA and FST results remained improved. Adverse events included cataracts and macular oedema.

Conclusions: preliminary results suggest that QR-110 has an acceptable safety profile, and potential for improvement of BCVA and light sensitivity.

Conflict of interest disclosure: The following conflict(s) of interest must be disclosed:
The authors received research support grants and travel grants from ProQR Therapeutics

Homozygous frameshift mutations in FAT1 cause a syndrome characterized by colobomatous-microphthalmia, ptosis, nephropathy and syndactyly

Brian Brooks1
Lahrouchi, Najim2; George, Aman1; Ratbi, Ilham3; Schneider, Ronen4; Elalaoui, Siham3; Moosa, Shahida5; Bharti, Sanita1; Sharma, Ruchi1; Abu-Asab, Mones1; Onojafe, Felix1; Adadi, Najlae3; Lodder, Elisabeth6; Laarabi, Fatima-Zahra7; Lamsyah, Yassine8; Elorch, Hamza9; Imane, Chebbar10; Postma, Alex11; Lougaris, Vassilios12; Plebani, Alessandro12; Altmueller, Janine13; Kyrieleis, Henriette14; Meiner, Vardiella15; McNeill, Helen16; Bharti, Kapil1; Lyonnet, Stanislas17; Wollnik, Bernd5; Henrion-Caude, Alexandra18; Berraho, Amina9; Hildebrandt, Friedhelm4; Sefiani, Abdelaziz3; Bezzina, Connie2
1. National Eye Institute, Bethesda, MD, USA
2. University Of Amsterdam, Amsterdam, Netherlands
3. Centre De Recherche En Genomique Des Pathologies Humaines, Rabat, Morocco
4. Dpt Pediatrics, Boston, MA, USA
5. Institute Of Human Genetics, Goettingen, Germany
6. Department Of Clinical And Experimental Cardiology, Amsterdam, Netherlands
7. Département De Génétique Médicale, Institut National D’hygiène, Agdal, Morocco
8. Service D’ophtalmologie B, Hôpital Des Spécialités, Agdal, Morocco
9. Service D ’ Ophtalmologie B, Hôpital Des Spécialités, Rabat, Morocco
10. Service D’ophtalmologie B,, Rabat, Morocco
11. Amsterdam UMC, Amsterdam, Netherlands
12. Pediatrics Clinic And Institute For Molecular Medicine, Brescia, Italy
13. Cologne Center For Genomics, Cologne, Germany
14. Department Of Pediatrics, Cologne, Germany
15. Department Of Human Genetics And Metabolic Diseases, Jerusalem, Israel
16. Department Of Developmental Biology, St. Louis, MO, USA
17. Laboratory Of Embryology And Genetics Of Human Malformation, Paris, France
18. Inserm UMR- 781, Département De Génétique, Paris, France

Purpose: To evaluate the role of the atypical cadherin, FAT1, in optic fissure closure and ocular malformation.

Methods: Exome sequencing of patients affected with coloboma, deep clinical phenotyping. Modeling in animal models (mouse, fish) and RPE cell culture.

Results: Five consanguineous families with four different homozygous presumed loss-of-function mutations in FAT1 were identified. Clinical findings include coloboma±microphthalmia; variable congenital blepharoptosis; skeletal abnormalities, including hypotrophy of digits and osseous syndactyly; and nephropathy. FAT1 is expressed in the developing optic cup and periocular mesenchyme of mouse. Knockout of Fat1/fat1a results in colobomatous microphthalmia in mouse and zebrafish embryos. FAT1 localized to the earliest cell-cell junctions in RPE, the initial cells to fuse during optic fissure closure, consistent with it having a role in the first stages of this developmental process.

Conclusions/Significance: Our findings establish FAT1 as a gene that, when mutated, results pleiotropic effects in human and in animal models. Our data suggest a role of FAT1 in forming the first contacts of presumptive RPE during optic fissure closure.

Conflict of interest disclosure: none

Molecular and cellular mechanisms in NR2E3-linked retinal degenerations

Pascal Escher
Inselspital, University Bern, Bern, Switzerland

Purpose: Description and analysis of mouse models of Nr2e3-linked retinal degenerations, and functional analysis of potential underlying molecular mechanisms

Methods: We characterized retinal degeneration in newly generated ‘knock-in’ mice with targeted modifications in the DNA-binding domain (DBD) and the ligand-binding domain (LBD) of Nr2e3; in vivo, by fundus photography, optical coherence tomography (OCT) and spectral electroretinography (ERG); post mortem by histology and immunohistochemistry. Additional in vitro analyses included RNA expression profiling (RNAseq), quantitative PCR, Western blotting and bioluminescence resonance energy transfer (BRET) assays.

Results: Knock-in mice homozygous for a targeted missense mutation in the LBD show a phenotype similar to the Nr2e3rd7/rd7 mice, an established mouse model of the recessively inherited retinal degeneration enhanced S-cone sensitivity syndrome (ESCS). Retinal spots and rosette-like structures first appear at postnatal day (P) 12 in the outer nuclear layer of the dorsal retina and reach maximal expansion at P21. At that age, the dorso-ventral M-cone gradient and the opposing ventro-dorsal S-cone gradient are present. Microglial cells and monocytes/macrophages are detected within ‘rosettes’. The highest density in rosettes is observed within a region located between 100 and 350 µM with respect to the optic nerve head where they persist the longest. Rosettes disappear by 9 to 12 months, and slow photoreceptor degeneration, at a rate of an approximately 3 % loss of outer nuclear layer thickness per month, is observed. The impact of mutations in the LBD on cofactor assembly was analyzed by BRET assays. Knock-in mice homozygous for a targeted missense mutation in the DBD exhibit a more severe phenotype. Remarkably, mice heterozygous for the DBD variant do not exhibit rosettes, but a thinning of the outer nuclear layer similar to the homozygous mice is observed. Finally, RNAseq analysis reveals distinct transcriptional regulation in the different mouse models.

Conclusion: The newly generated knock-in mouse models allow to delineate the contribution of the Nr2e3 DBD and the LBD to retinal degeneration.

Conflict of interest disclosure: The following conflict(s) of interest must be disclosed:
Pascal Escher received honorary or consultation fees from Novartis Pharma, Switzerland

ATOH7 loss-of-function mutations in a family with hypoplasia of the optic nerve

David Grubich Atac1
Koller, Samuel1; Hanson, James2; Feil, Silke1; Tiwari, Amit1; Bahr, Angela1; Baehr, Luzy1; Magyar, Istvan1; Kottke, Raimund3; Gerth-Kahlert, Christina2; Berger, Wolfgang1
1. Institute Of Medical Molecular Genetics, University Of Zurich, Schlieren, Switzerland
2. Department Of Ophthalmology, University Hospital Zurich, Zurich, Switzerland
3. Department Of Diagnostic Imaging, University Children’s Hospital Zurich, Zurich, Switzerland

Purpose: Bilateral optic nerve hypoplasia (ONH) is a congenital optic nerve abnormality due to underdevelopment of retinal ganglion cells (RGCs), often resulting in legal blindness. Mutations in several genes have been associated to this disease, however more than half of all cases remain without a clear molecular explanation. The early retinal transcription factor atonal basic helix-loop-helix (bHLH) transcription factor 7 (ATOH7) is expressed in retinal progenitors and has a key role in RGC differentiation. Consequently, ATOH7 is a potential candidate gene for ONH. Here we present two siblings with bilateral ONH and macular hypoplasia where exome sequencing identified compound heterozygous missense variants in ATOH7, one very rare and one novel, affecting a conserved residue within the HLH domain. The variants were predicted deleterious by several algorithms. In addition, we have functionally characterized the identified variants at the protein level.

Methods: Whole exome sequencing (TruSeq exome) of the index patient was performed with an Illumina NextSeq 550 platform and segregation analysis of putative pathogenic variants in additional family members by Sanger sequencing. Expression constructs with ATOH7 patient-derived variants were cloned into a plasmid vector for functional characterization by transgene expression in HEK293T cells. To stimulate heterodimerization, a putative dimerization partner (E47) was cloned and co-expressed. ATOH7 patient variants were characterized by Western blot and cycloheximide chase assays, ELISA based protein and DNA interaction assays as well as luciferase reporter assays.

Results: Protein amount as well as stability of ATOH7 variants were significantly reduced in the presence of a putative dimerization partner E47. Functional assessment showed significant reduction in heterodimerization as well as DNA-binding of the two ATOH7 variants. Finally, transcriptional activation of luciferase reporter expression was abolished.

Conclusions: Increased dimerization-dependent protein degradation as well as structural changes in dimerization and chromatin binding ultimately cause loss of function of the transcription factor ATOH7 with the two patient-derived variants. These findings strongly support pathogenicity of the two ATOH7 sequence variations. Additionally, this report highlights the impact of altered ATOH7 dimerization on protein amount and function. Genetic analysis of this gene should be performed in patients with ONH.

Conflict of interest disclosure: none

Prevention of intravitreal melphalan-induced chorioretinopathy: identification of potential risk factors

Francis Munier
Gaillard, Marie-Claire; Bergin, Ciara; Houghton, Susan; Stathopoulos, Christina
Jules-Gonin Eye Hospital, Lausanne, Switzerland

Purpose: retinal toxicity of melphalan is a potentially sight-threatening complication of intravitreal chemotherapy. Identification of risk factors for the complication remains as yet inconclusive.

Methods: retrospective review of 90 consecutive eyes receiving their primary treatment for vitreous disease in Jules-Gonin Eye Hospital with intravitreal melphalan injections between 2008 and 2017 according to the safety-enhanced technique. Melphalan-induced retinopathy was assessed based on the previously described grading of the toxicity. For each case, timing and total injected dose until the apparition of the first clinical signs of the complication were noted. Mean total intravitreal concentration at time of the complication was evaluated without and with consideration of the intraocular tumor volume. Vitreous volume calculation was based on axial length estimated according to age or corrected age in case of prematurity. Tumor volume was estimated based on height, longitudinal and transverse radii of each tumor measured with ultrasonography (12 MHz).

Results: Overall 37 eyes (n=37/90, 41%) suffered a melphalan-induced chorioretinopathy, which was grade 1 in 15 (n=15/90, 17%), grade 2 in 19 (n=19/90, 21%) grade 3 in 3 eyes (3/90, 3%). There were no significant differences between the eyes presenting grade 1 or 2 versus grade 3 toxicity regarding the mean time to develop the complication, the total injected dose until the first clinical signs, or the injected concentration when the latter was evaluated taking into account the age-related vitreous volume only. On the contrary, when the intravitreal concentration was calculated substracting the tumor volume from the age-related vitreous volume, the toxic retinopathy was significantly related to the intravitreal drug concentration when exceeding 10µg/ml (p=0.04).

Conclusion: our findings imply that in addition to age-matched vitreous volume, intraocular tumor volume is an important factor to consider when choosing the melphalan dose to be injected into the vitreous. Recommended melphalan doses for intravitreal chemotherapy according to age and percentage of the vitreous volume occupied by the tumor(s) are presented.

Conflict of interest disclosure: none

The genetic and clinical landscape of nanophthalmos in an Australian cohort

Jonathan Ruddle1
Souzeau, Emmanuelle2; Awadalla, Mona S.2; Burdon, Kathryn3; Owen, Siggs2; Craig, Jamie2
1. Royal Childrens Hospital, Parkville, Australia
2. Department Of Ophthalmology, Flinders University, Flinders Medical Centre, Bedford Park, SA, Australia
3. Menzies Institute For Medical Research, Hobart, TAS, Australia

Purpose: Refractive error is caused by a disparity between the axial length and focusing power of the eye. Microphthalmia is a rare ocular abnormality in which one or both eyes are abnormally small, typically causing hypermetropic refractive error. Nanophthalmos, is a rare subtype of microphthalmia associated with an increased risk of glaucoma. These findings detail the genetic architecture of nanophthalmos in a predominantly European cohort, their relative clinical phenotypes, and highlight the shared genetic architecture of rare and common disorders of refractive error.

Methods: Genetic analysis of cohort of 19 unrelated nanophthalmic probands from the Advanced Glaucoma registry. Correlated with clinical findings in the proband and affected family members.

Results: Nine probands (47.4%) were assigned a genetic diagnosis, with variants in PRSS56, MFRP, and previously reported variants in TMEM98 and MYRF. Four probands were explained by biallelic variants in PRSS56, encoding a secreted serine protease important for prenatal and postnatal ocular growth. Two of the four PRSS56 probands harboured the previously described c.1066dupC frameshift variant, yet their surrounding haplotypes were distinct from each other, and from a previously reported Tunisian c.1066dupC haplotype. Three probands had biallelic variants in MFRP. Mean axial length was shorter in the subset of individuals with a genetic diagnosis compared to those without, with PRSS56 variants associated with the shortest axial length.

Conclusions/Significance: A large proportion of nanophthalmos can be explained by the known and recently described genes. Clinically those with a gene result had shorter axial length, lower acuity, higher hypermetropia and earlier glaucoma.

Conflict of interest disclosure: none

Pedigree analysis of familial primary concomitant horizontal strabismus in a south Asian population

Zia Chaudhuri1, 2
Ganguly, Sourav2; Debnath, Pinaki3
1. Lady Hardinge Medical College & Associated Hospitals, University Of Delhi, New Delhi, India
2. Vision Research Lab, PGIMER, DR RML Hospital, New Delhi, Delhi, India
3. Department Of Pediatric Surgery, PGIMER, DR RML Hospital, New Delhi, New Delhi, India

Purpose: Primary concomitant strabismus (PCS) comprising intermittent exotropia (IE) and accommodative esotropia (ET) are two most common forms of ocular misalignment. Familial forms of PCS have been observed across all populations. However, in most cases, neither a definitive mode of inheritance nor other genetic and epigenetic determinants are concretely established. Recently next generation sequencing (NGS) technology has emerged as a powerful tool in discovery genomics and a large number of novel disease-causing variants are being reported. As a first step, we recruited informative families for subsequent genetic analysis for disease-causing variant identification.

Methods: All consecutive families of south Asian origin living in different parts of India, with two or more affected members with PCS were prospectively recruited at the ophthalmic outpatients department (OPD) of Lady Hardinge Medical College and PGIMER, Dr RML Hospital, New Delhi, India from August 2014 to April 2019. Detailed phenotypic evaluation and pedigree documentation by Cyrillic 3.0.400 software was performed.

Results: Of the 62 recruited PCS families, 100% concordance was observed in 56, 24 with ET and 32 with XT. In 4 families, the affected members demonstrated both, ET and XT. 15/24 ET (62.5%), 26/32 XT (81.2%) and 3 of 4(75%) families with both ET and XT demonstrated the phenotype of PCS in at least two generations implying possible vertical transmission. In 2 families, one of the siblings each had XT and the other sibling demonstrated Duanes retraction syndrome (DRS). Thus while these families were considered as having familial strabismus, there was coexistence of comitant XT in both cases alongwith DRS, esotropic in one and exotropic in the other. Consanguinity was observed in 2 families, both with ET. In two families with XT and autosomal recessive (AR) inheritance, nystagmus and low vision were additional phenotypes. In another two families with XT, the proband had autism in one and an associated mitochondrial myopathy in the other.

Conclusions: The pedigree analysis of this large and unique familial cohort in this geographical location of south Asia, recruited for future genetic analysis, opens up perspectives on the varied phenotypic heterogeneity of the condition in this population

Conflict of interest disclosure: none

Session 3 – Stem Cells

Treatment options for limbal stem cell deficiency in inhereted eye diseases

Björn Bachmann
Department Of Ophthalmology, Cologne, Germany

A wide range of diseases can lead to limbal stem cell deficiency (LSCD). Besides conservative treatment options for the first time in Europe there is an approved surgical therapy for the transplantation of autologous limbal stem cells available. However, autologous limbal stem cells are availble in unilaterally affected patients only and genetic causes of LSCD typically affect both eyes. Transplantation of allogeneic stem cells often leads to graft failure within the first few years despite systemic immunosuppression. An alternative surgical approach is the implantation of a Boston type I keratoprosthesis (BKPro). Although the initial results after implantation of a BKPro are very exciting the patients are confronted with a wide range of complications during the postoperative course. Recent experimental approaches aim to transplant allogeneic epithelial stem cells with less immunogenic properties.

Conflict of interest disclosure: none

Differentiation and characterization of RPE from hiPSC and its subretinal transplantation in RCS rats

Rajani Battu
Pal, Rajarshi
Eyestem Research Private Ltd, Bangalore, India

Purpose: Retinal degenerative diseases like Age-related Macular Degeneration (AMD) and Retinitis Pigmentosa (RP) cause irreversible retinal cell death and progressive vision loss in millions of people across the world. Subretinal transplantation of cells is a promising experimental method for their treatment. Here, we tested the efficacy of transplantation of (Eyecyte-RPE) RPE cells to rescue photoreceptors and visual function in the RCS rat model of retinal degeneration.

Methods: We have successfully generated retinal pigment epithelial (RPE) cells from human iPSC and characterized them wrt their identity, purity and potency. Transplantation of 50K or 100K cells into the subretinal space was performed as a suspension in RCS rats; BSS+ served as controls. Optomotor tracking was used to measure visual acuity of all animals. Half the animals were sacrificed at P60 and the other half at P90.

Results: All the experimental animals showed successful delivery of cells and bleb formation. Optomotor tracking thresholds were rescued in each cell treated dose groups over that of control animals. Histological analysis revealed significant photoreceptor protection above that of controls at both sacrifice ages in both cell dose groups. Quantification of cone counting revealed a significantly higher number of cones in cell treated groups compared with control eyes, most evident at P90. Approximately 10% of the transplanted RPE cell markers expressed Ki67, a cell proliferation marker.

Conclusion: When transplanted into the subretinal space of RCS rats, Eyecyte-RPE delayed the loss of visual acuity in the RCS rat over that of controls at all ages tested. Rod and cone photoreceptors were rescued in the area of the grafts for up to 70 days post-transplantation. RPE transplantation is a promising treatment for degenerative retinal diseases.

Conflict of interest disclosure: The following conflict(s) of interest must be disclosed:
Rajani Battu is/are stock shareholder of Eyesystems Research Private LTD

Proteomics profiling of retinoblastoma derived exosomes

Angela Galardi1
Colletti, Marta1; Lavarello, Chiara2; Di Paolo, Virginia1; Mascio, Paolo1; Russo, Ida1; Cozza, Raffaele1; Romanzo, Antonino3; Valente, Paola3; De Vito, Rita4; Pascucci, Luisa5; Peinado, Hector6; Montero Carcaboso, Angel7; Munier, Francis8; Petretto, Andrea2; Locatelli, Franco1; Di Giannatale, Angela1
1. Department of Pediatric Hematology/Oncology, Bambino Gesù Children’s Hospital, IRCCS, Rome, Italy
2. Core Facilities-Proteomics Laboratory, IRCCS Istituto Giannina Gaslini, Genoa, Italy
3. Ophtalmology Unit, Bambino Gesù Children’s Hospital, IRCCS, Rome, Italy
4. Department of Pathology, Bambino Gesù Children’s Hospital, IRCCS, Rome, Italy
5. Department of Veterinary Medicine, University of Perugia, Perugia, Italy
6. Microenvironment & Metastasis Group, Molecular Oncology Program, Spanish National Cancer Research Centre (CNIO), Madrid, Spain
7. Institut De Recerca Sant Joan De Deu, Barcelona, Spain
8. Hopital Ophtalmique Jules Gonin – Fondation Asile Des Aveugles, Lausanne, Switzerland

Purpose: Retinoblastoma (RB) is the most common intraocular cancer of childhood. Despite recent advances in conservative treatment have greatly improved the visual outcome, local tumor control remain difficult in presence of massive vitreous seeding. Thus, the identification of new biomarkers is crucial to design more effective therapeutic approaches. Traditional biopsy has long been considered unsafe in RB, due to the risk of extraocular spread. Exosomes, nano-sized vesicles containing nucleic acids and proteins, represent an interesting alternative to detect tumor-associated biomarkers. The aim of this study was to determine the protein signature of exosomes derived from RB tumors (RBT) and vitreous seeding (RBVS) primary cell lines.

Methods: Exosomes from 4 RBT (HSJD-RBT1, HSJD-RBT2, HSJD-RBT5, HSJD-RBT14) and 3 RBVS (HSJD-RBVS1, HSJD-RBVS3, HSJD-RBVS10) cell lines were isolated by high speed ultracentrifugation. Vesicles number and size were confirmed by NanoSight and Scanning Electron Microscopy. Protein content was analyzed by bicinchonic-acid assay and high resolution mass spectrometry.

Results: A total of 5404 proteins were identified. Among those, 1940 and 409 were exclusively expressed in exosomes from at least one RBT and one RBVS respectively. 3055 proteins were in common between the two groups. Gene Ontology analysis identified proteins primarily involved in unfolded protein response, metabolic processes and cellular response to hypoxia in both groups. Only in RBT-exosomes were found proteins involved in neuronal development, vesicular traffic, oxidative metabolism, ion channels and transport. Lastly, we found 15 proteins exclusively expressed in all RBT-derived exosomes (e.g SNAP25, NCAN, ABI2) and 6 proteins in RBVS-derived exosomes (e.g integrin beta-3, PSMB6, GALK1, CREBBP).

Conclusions: This work suggests that the proteomic profiling of RB-derived exosomes could reflects the signature of the primitive tissue and may be considered as potential tumor biomarkers.

Conflict of interest disclosure: none

Franceschetti Medal & Lecture 2019

Franceschetti Medal Introduction

Franceschetti Medal & Lecture 2019

Clinical features and molecular basis of X-linked retinoschisis: From mechanism to therapy

Paul Sieving
National Eye Institue, NIH, Bethesda, MD, USA

Purpose: To describe X-linked retinoschisis disease (XLRS) and our gene therapy treatment efforts. XLRS is a monogenic retinal dystrophy with a complex phenotype that includes retinal structural defects resulting in schisis cavity formation. A prominent feature of XLRS disease historically has been the selective reduction of the electroretinogram b-wave but not a-wave. While the b-wave loss frequently is considered a curiosity, it signifies a fundamental facet of XLRS disease, namely a failure of synaptic transmission from photoreceptors to the depolarizing bipolar cells (DBCs).

Methods: We studied the molecular basis of XLRS pathology and are conducting a gene therapy trial.

Results: In probing the molecular basis of this condition, we found that the retinoschisin RS1 molecule is required for proper localization of signaling molecules in the post-synaptic dendritic inputs to the DBCs. The absence of RS1 protein in XLRS disease results in deficient visual signal transmission despite normal photoreceptor function. Hence the XLRS phenotype overlaps Congenital Stationary Night Blindness (CSNB). Providing normal RS1 protein by gene therapy restores the b-wave in the RS1-KO mouse model and, if extended to the human condition, presumably will at least partially correct the visual failure. We initiated a Phase I/IIa human XLRS gene therapy trial (Feb 2015; ClinicalTrials.Gov NCT02317887). Our clinical scAAV8-hRS/IRBP-RS1 vector contains the human RS1 cDNA and a tissue-specific RS1 promoter plus an IRBP enhancer. Pre-clinical studies indicated that “a fully normal level of RS1 expression” was not necessary for therapeutic effect in the XLRS mouse. We have dosed eleven subjects at 1e9 to 3e11 vg/eye. The primary outcome is safety, and secondary outcomes include monitoring for treatment benefit, with visual acuity and fields, ERG responses and retinal structure by OCT imaging. In one subject dosed at 1e11 vg/eye, we observed complete closure of macular schisis cavities two weeks later. We are dosing additional subjects and evaluating possible functional benefits.

Discussion: Currently we are exploring methods to ameliorate the ocular inflammatory response we observe following intravitreal administration of the vector, while monitoring the formation of anti-AAV and anti-RS1 antibodies. Results will be described.

Conflict of interest disclosure: None

Session 4 – Biomarkers for Substantiating Success in Treatment

AAV2-hCHM subretinal delivery to the macula in choroideremia: Performance of outcome measures

Tomas Aleman1
Huckfeldt, R.M.3; Serrano, L.W.1; Vergilio, G.1, 2; Pearson, D.J.1, 2; Uyhazi, K.E.1, 2; McCague, S.4; Chung, D.5; Liu, E.5; Morgan, J.I.W.1, 2; Pierce, E.A.3; Eliott, D.3; Bennett, J.1, 2; Comander, J.3; Maguire, A.M.1, 2, 4
1. Scheie Eye Institute at the Perelman Center for Advanced Medicine and the Center for Advanced Retinal and Ocular Therapeutics, Department of Ophthalmology, University of Pennsylvania, Philadelphia, PA,
2. Massachusetts Eye and Ear, Department of Ophthalmology, Harvard Medical School, Boston, MA,
3. The Children’s Hospital of Philadelphia, Philadelphia, PA,
4. Spark Therapeutics, Philadelphia, PA

Purpose: To assess preliminary safety and efficacy data of the investigational subretinal delivery of a recombinant adeno-associated virus serotype 2 (AAV2) vector carrying a human REP1-encoding cDNA in choroideremia (CHM).

Methods: Ten subjects with CHM (ages 26-57 years at injection), received uniocular subfoveal injections of low dose (up to 5×1010 vector genome (vg) per eye, n=5) or high dose (up to 1×1011 vg per eye, n=5) AAV2-hCHM. Patients were evaluated pre- and post-operatively at study-defined follow up visits for 2 years. Ocular safety was assessed by ophthalmic examination, perimetry, spectral domain optical coherence tomography (SD-OCT) and short-wavelength autofluorescence (SW-FAF) and by conventional automated perimetry and microperimetry.

Results: There were no surgery-related complications or unexpected adverse events. By two years visual acuity (VA) returned to baseline in all but one patient who slowly recovered to -17 letters of baseline. With the exclusion of this patient, mean VA letter counts differences (2 year minus baseline) were similar in injected (-1.7 letters) compared to uninjected (-0.3 letters) eyes. Two patients showed greater VA counts (5-6 letters) in the injected eye compared to baseline and to the uninjected control. Mean sensitivity by microperimetry changed minimally in injected (mean±SD=-0.7±0.75dB) and uninjected (-0.3±0.59 dB) eyes. There were no significant differences between injected and uninjected eyes in absolute dark-adapted cone-mediated sensitivities at the fovea or within the central 30° of eccentricity. There was a slightly slower mean rate of reduction of the IS/OS band horizontal extent in injected eyes (-69±59μm/year) compared to uninjected eyes (-96±67μm/year), which corresponded in extent to the areas of SW-FAF. There were no obvious dose-dependent relationships.

Conclusions: VA in 9/10 subjects was unchanged (less than ±10 letters difference from baseline) after the subfoveal injections of AAV2-hCHM and in uninjected eyes at 2 years of follow-up. Acute (~72 hours) localized foveal thinning and slow, partial recovery of VA in one patient suggests non-vector related individual vulnerability to the subfoveal injection. Residual islands of relatively preserved retina continued to shrink in both injected and uninjected eyes. Longer observation intervals are required to better evaluate the significance of these observations.

Support: Spark Therapeutics Clinical Trials Agreement, National Institutes of Health (NEI-K12EY015398-10, NIH R01EY028601), Research to Prevent Blindness, Foundation Fighting Blindness, Hope for Vision, Macula Vision Research Foundation, the Paul and Evanina Bell Mackall Foundation Trust, and The Pennsylvania Lions Sight Conservation and Eye Research Foundation.
Clinical Trial Registration: NCT02341807

Conflict of interest disclosure: The following conflict(s) of interest must be disclosed:
F Spark Therapeutics (AMM); P (JB); S GenSight Biologics, Spark Therapeutics (JB); C Sanofi (JB); F Biogen, Limelight Bio (JB). Jason – Beam therapeutics, Sanofi, Editas Medicine, Gensight, Blue Cross Blue Shield (Consultant for all) Eric – Spark (F)

High-resolution retinal imaging analysis in female carriers of choroideremia

Kiyoko Gocho
Nippon Medical School, Chiba Hokusoh Hospital, Inzai, Japan

Purpose: Choroideremia is an X-linked disease that causes degeneration in choriocapillaris, retinal pigment epithelium (RPE) and retinal photoreceptors. Heterozygous female carriers of choroideremia typically do not have any visual symptoms but often have severe funduscopic changes. The purpose of this study was to characterize the cone photoreceptor mosaic and choroidal morphology of these carriers.

Methods: This research was a clinical case series study. Six genetically identified female carriers of choroideremia from four different families were included. Each patient underwent a comprehensive ophthalmic examination, including: full-field electroretinography (ERG), color fundus photography, subfoveal choroidal thickness (SFCT) measured with spectral-domain optical coherence tomography (OCT), and cone photoreceptor density assessed using an adaptive optics (AO) retinal camera at temporal eccentricities ranging from 2 to 8 degrees. SFCT and cone density findings were compared to control data previously obtained in a group of healthy subjects.

Results: Carriers’ age ranged between 10 and 66 years. Best-corrected visual acuity was equal to or higher than 20/20. With conventional fundoscopy, severe retinal depigmentation was observed in one carrier and scattered depigmentation was present in 3 other cases. However, in all patients, SFCT was within normal limits, and cone photoreceptor density values measured with AO imaging did not significantly differ from those found in healthy controls.

Conclusions: The findings indicate that despite the presence of distinctive depigmentation of the retinal pigment epithelium in female carriers of choroideremia, their cone photoreceptor density and SFCT are well-preserved. Therefore, unlike conventional fundoscopy, both OCT and AO imaging provided objective assessments that were consistent with visual performance findings.

Conflict of interest disclosure: The following conflict(s) of interest must be disclosed:
Kiyoko Gocho received grants or research support from Kaken Grant type(C) No.18K09426
Spouse of Kiyoko Gocho is CEO of Imagine eyes

Multimodal imaging of patients with Best Vitelliform Macular Dystrophy (BVMD): a 4-year follow-up study

Jose Ronaldo Lima de Carvalho Jr1, 2
Ragi, Sara2; Park, Karen Sophia2; Sparrow, Janet R.2; Maumenee, Irene2; Tsang, Stephen H.2
1. Federal University Of Pernambuco, Cidade Universitaria, Recife, Brazil
2. Columbia University, New York, NY, USA

Purpose: To test the hypothesis that imaging biomarkers can be used to measure outcome in upcoming Best Vitelliform Macular Dystrophy (BVMD) treatment trials.

Methods: A retrospective analysis of 31 patients (59 eyes) from 27 families with a clinical and genetic diagnosis of dominant BVMD was performed. Three eyes were excluded from analysis due to poor image quality. SD-OCT, SW-AF and NIR-AF images were taken at the same visit. A second set of imaging was performed in 15 patients (30 eyes). The diameter of the lesion measured by the two different autofluorescence techniques were correlated with the measurements made by SD-OCT. Central macular thickness, foveal height of the lesion and foveal outer nuclear layer (ONL) thickness were measured by SD-OCT. Likewise, ONL thickness at temporal (T-ONL) and nasal (N-ONL) limits of the lesion and at 500 µm from the border of the lesions (5T-ONL and 5N-ONL) towards the healthy retina were evaluated. In addition, the area of the macular lesion was manually measured on both SW-AF and NIR-AF. Comparative statistics was used to calculate differences between the calculated means. The Pearson correlation coefficient was used to evaluate the relationships between each imaging modality.

Results: Among 59 eyes, one eye classified in the pre-vitelliform stage did not exhibit a lesion after 2 years of follow-up but revealed a hypoautofluorescent signal on NIR-AF that was not observed on SW-AF. The mean follow-up time was 4.11±0.54 years. Significant positive correlations were found among SD-OCT, SW-AF, and NIR-AF when used to measure lesion diameter (P<0.001). Distinct regions of the lesions, namely T-ONL, N-ONL, 5N-ONL, decreased in thickness by -3.83±2.26 µm/year, -5.03±2.01 µm/year, -5.11±2.69 µm/year, respectively, over time. No progression was observed in the diameter and area of the lesion as measured by each imaging modality.

Conclusion: NIR-AF appears to have greater sensitivity to the early pre-vitelliform stage in BVMD. As significant changes were observed in the ONL of the lesions over time, our data suggests that ONL measurements may be used as an anatomical outcome measure for clinical trials. Future studies should include parameters such as microperimetry, which may additionally serve as a form of functional outcome measure for BVMD.

Conflict of interest disclosure: none

Characterization of the Brazilian ARSACS phenotype: clinical, ophthalmological, neuroimaging, and genetic features of fourteen cases

Juliana Sallum1
Rezende Filho, Flavio2; Pedroso, Jose Luiz2; Kok, Fernando2; França Junior, Marcondes4; Vasconcelos, Ingrid4; Giunti, Paola5; Barsottini, Orlando3; Marques, Wilson6; Lourenço, Charles6
1. Ophthalmology and Visual Sciences Department, Universidade Federal de São Paulo, Brazil
2. Ataxia Unit, Department of Neurology, Universidade Federal de São Paulo, Brazil
3. University of São Paulo, Brazil
4. University of Campinas, Brazil
5. UCL Institute of Neurology, London, UK
6. University of São Paulo – Ribeirão Preto, Brazil

Background: Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is a rare, progressive neurodegenerative condition described in Charlevoix-Saguenay region, where it causes spasticity, ataxia and peripheral neuropathy (1). Retinal nerve fiber layer thickening is present in most Canadian cases (1,2). Cerebellar atrophy and linear pontine hypointensities on MRI support diagnosis (3). More than 200 mutations in the causative gene, SACS, have been reported throughout the world, and disctinct ARSACS features occur in non-Canadians individuals. Phenotype characterization is important to understand molecular pathogenesis and improve accuracy in diagnosis.

Methods: Magnetic resonance imaging of the skull, retinography and optic coherence tomography (OCT) were performed in fourteen consecutive cases of genetically confirmed ARSACS. Detailed neurological history and examination were recorded. We obtained informed consent from all patients, and approval by Ethics Committee.

Results: We included 14 patients (10 females) from 10 families (age range 16-57 years). Age at onset was within the first decade in twelve cases, 11 years in one and 44 in another. All had ataxia, peripheral neuropathy and spasticity. One had learning disability and psychosis at the age of 23. Retinal nerve fiber hypertrophy in OCT was seen in 12/13. Cerebellar atrophy (14/14), biparietal atrophy (13/14), and linear pontine hypointensities (13/14) were the most consistent radiological signs. Genetic analysis revealed 16 different SACS gene mutations and thirteen novel variants.

Conclusions: Retinal and neuroimaging changes are common feature in Brazilian ARSACS. OCT and MRI may provide useful clues during diagnostic work-up of spastic ataxias.

Conflict of interest disclosure: none

Giessen 2019 – Day 1 Photo Gallery