Ceramide synthase TLCD3B is a novel gene associated with human recessive retinal dystrophy Renae Elaine Bertrand B.S.,1,2 Jun Wang Ph.D.,2,3 Kaitlyn H. Xiong B.S.,2,4 Chinthana Thangavel,2,4 Xinye Qian B.A.,1,2 Rola Ba-Abbad FRCS Ph.D.,5 Qingnan Liang M.S.,1,2 Renata T. Simões,6 Shirley A. M. Sampaio,7 Keren J. Carss Ph.D.,8,9 F. Lucy Raymond M.D. Ph.D.,8,10 Anthony G. Robson M.Sc. Ph.D.,11 Andrew R. Webster FRCOphth,5,11 Gavin Arno Ph.D.,5,11,12 Fernanda Belga Ottoni Porto M.D. Ph.D.,7 Rui Chen Ph.D.2,3,* 1 Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX 77030, USA 2 Human Genome Sequencing Center, Baylor College of Medicine, Houston, TX 77030, USA 3 Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA 4 Department of BioSciences, Rice University, Houston, TX 77005, USA 5 Moorfields Eye Hospital, London, EC1V 2PD, UK 6 Instituto de Ensino e Pesquisa da Santa Casa de Belo Horizonte (IEP/SCBH), Minas Gerais, Brazil 7 INRET Clínica e Centro de Pesquisa, Belo Horizonte, Minas Gerais, Brazil 8 Department of Haematology, University of Cambridge, Cambridge, UK 9 NIHR BioResource-Rare Diseases, Cambridge University Hospitals, Cambridge Biomedical Campus, Cambridge, UK 10 Department of Medical Genetics, Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK 11 UCL Institute of Ophthalmology, London, EC1V 9EL, UK 12 Great Ormond Street Hospital for Children, London, UK * Rui Chen, 713-798-5194, [email protected] 1 ABSTRACT Purpose Previous studies suggest that ceramide is a proapoptotic lipid as high levels of ceramides can lead to apoptosis of neuronal cells, including photoreceptors. However, no pathogenic variant in ceramide synthases has been identified in human patients and knockout of various ceramide synthases in mice has not led to photoreceptor degeneration. Methods Exome sequencing was used to identify candidate disease genes in patients with vision loss as confirmed by standard evaluation methods, including electroretinography (ERG) and optical coherence tomography. The vision loss phenotype in mice was evaluated by ERG and histological analyses. Results Here we have identified four patients with cone-rod dystrophy or maculopathy from three families carrying pathogenic variants in TLCD3B. Consistent with the phenotype observed in patients, the Tlcd3bKO/KO mice exhibited a significant reduction of the cone photoreceptor light responses, thinning of the outer nuclear layer, and loss of cone photoreceptors across the retina. Conclusion Our results provide the first link between loss-of-function variants in a ceramide synthase gene and human retinal dystrophy. Establishment of the Tlcd3b knockout murine model, the first in vivo photoreceptor cell degeneration model due to loss of a ceramide synthase, will provide a unique opportunity in probing the role of ceramide in survival and function of photoreceptor cells. Retinal degeneration; ceramide synthase; cone-rod degeneration; novel disease gene; TLCD3B 2 INTRODUCTION Inherited retinal dystrophies (IRDs) are a diverse set of disorders whose primary site of pathology is often, but not exclusively, the rod and/or cone photoreceptors. Causative genes vary in their involvement of each disorder; at least 250 genes have been identified to underlie retinal dystrophy (https://sph.uth.edu/retnet). The advances in next-generation sequencing and its applications for molecular diagnostics for individuals with IRDs have increased the diagnostic yield up to 70%1. Identifying the molecular diagnosis in these cases assists in clinical management of patients, and future inclusion in therapeutic trials. Clinical investigation involving detailed imaging and electrophysiology in humans, in concert with in-depth phenotyping of relevant animal models can provide insights into the basic pathophysiology. There is clinical heterogeneity within and overlap between different IRDs. Cone-rod dystrophy (CRD) and maculopathy are two broad diagnoses that encompass varying phenotypes and it is possible for a patient’s diagnosis to change as the disease progresses. CRD and maculopathy are IRDs characterized by a shared clinical presentation of reduced central vision and significant peripheral cone dysfunction more than rod system dysfunction evident on electroretinography (ERG), indicating significant cone photoreceptor involvement in the pathology2 3. It has been reported that homeostasis of ceramide plays an important role in retinal neuron survival. Ceramides are a class of sphingolipids that are abundant in cell membranes and serve as important second messengers. Ceramide is generated through four pathways, de novo synthesis, the salvage pathway, the sphingomyelinase pathway, and the exogenous ceramide- recycling pathway, where ceramide synthases participate in all but the sphingomyelinase pathway4 5. In mammals, six ceramide synthases (CERS), CERS1-6, each regulate the formation of a specific set of ceramides, which mediate distinct functions5. There is redundancy in the ceramides synthesized by each synthase, but no ceramide synthase can completely compensate for another6-10. Ceramides have been shown to mediate many cell-stress responses, including apoptosis5 11-13. Interestingly, both high and low levels of ceramides have been linked to apoptosis. Long-chain ceramides are known to be pro-apoptotic in many contexts, including the 3 retina and brain11-15. A reduction in ceramide levels has been previously linked to neurodegeneration, specifically of Purkinje cells in the cerebellum of CerS1 deficient mice10. So far, pathogenic variants in ceramide synthases have been associated with diseases, such as epilepsy6 16 17, but not with retinal degeneration in patients, and previous ceramide synthase knockout mouse models have not shown retinal degeneration phenotypes18. Previous studies have identified Ceramide Kinase Like (CERKL) to be a retinal degeneration gene19, but it has been established that CERKL does not participate in sphingolipid metabolism20 21. In addition to the canonical ceramide synthases, it has been reported that the protein encoded by the TRAM/LAG1/CLN8 Domain 3B (TLCD3B, formally known as FAM57B) gene also has ceramide synthase activity. An in vitro study showed that TLCD3B can function as a novel ceramide synthase and is responsible for synthesis of C16-, C18-, and C20-ceramides22. The in vivo function of TLCD3B has not yet been confirmed and its connection with the central nervous system has not yet been investigated. While it has been established that maintenance of ceramide levels is important for retinal cell survival, the roles of ceramides and ceramide synthases are not well studied in the retina. In this study, we have identified pathogenic variants in TLCD3B in four patients from three unrelated families diagnosed with CRD or maculopathy. Consistent with the patient phenotype, disruption of Tlcd3b in mice leads to cone photoreceptor degeneration. Our results provide the first direct link between defects in a ceramide synthase and human retinal degeneration. MATERIALS AND METHODS Ethics Statement Informed consent was acquired from all of the Brazilian patients and participating family members who chose to participate. All of the procedures were approved by appropriate institutional review boards and ethics committees (Santa Casa de Belo Horizonte Research Ethics Committee CAAE: 11168613.0.0000.5138), and adhered to the tenets of the declaration of 4 Helsinki. For Patient C.IV.3 (GC18125), all diagnostic procedures were approved by the UK Research Ethics Committee, granted by the Cambridge South REC, informed consent was obtained from all patients or guardians, and procedures adhered to the tenets of the declaration of Helsinki. All animal procedures and experiments conformed to the IACUC-approved protocol. Bioinformatics Analysis Patients A.II.1, A.II.2 and B.II.1 were subjected to exome sequencing. After exome sequencing, BWA v.0.7.1223 was used to map the reads to the human genome assembly hg19. The retinal disease-causing variants were identified as previously described24. Details of the analysis are available in the supplemental methods. Patient C.IV.3 was subjected to genome sequencing, which was carried out as part of the NIHR BioResoirce – Rare Disease study as previously descibed25. Rare variant interrogation was performed using a curated virtual gene panel of known retinal dystrophy genes to exclude a known cause of disease. Following this, rare, biallelic loss of function variants in genes not harbouring homozygous null alleles in the ExAC dataset were interrogated25. Variant filtering and prioritization were performed as previously described25. Clinical Evaluation All Brazilian patients and family members have been examined by the same care provider (author FBOP), including best-corrected visual acuity (BCVA), slit-lamp biomicroscopy, dilated-pupil fundus examination, cycloplegic refraction, optical coherence tomography (OCT) (Cirrus HD-OCT, Carl Zeiss Meditec, Germany), fundus imaging, fundus autofluorescence (FAF) imaging (Topcon retinal camera TRC-NW8, Topcon Co., Japan), and full-field ERG (ffERG) (UTAS Sunburst, LKC Tecknologies, USA). 5 Patient C.IV.3 (GC18125) underwent examinations at the inherited Eye Disease clinics at Moorfields Eye Hospital, London, UK, which included BCVA, dilated fundus examination, OCT (Spectralis HRA +OCT, Heidelberg Engineering, Germany), FAF imaging (Spectralis HRA +OCT, Heidelberg Engineering, Germany),