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Before sharing a summary of our inaugural fundraiser, we again want to thank all who joined us on Saturday, April 24th. It was such a fantastic day. Well beyond our expectations. What was our Spring Fundraiser? It was a virtual walk/run/bike that encouraged participants to find the most unique location and take a picture with their race bib. The idea: raise awareness about RP, raise funds for medical research, and "show someone with RP the world". For this inaugural event, Cate was the face on every race bib. For future Spring Fundraisers, RP Hope will feature others affected by RP. For example, registered participants will walk or run or bike, hike, or kayak and "show" the world to someone like Lance Johnson, Molly Burke, or Harriette Hubbard. Making this a Virtual Event Originally, we planned to host two events; one in the U.S. and one in Europe, targeting the two time zones in which most of our friends and family lived. But, friends from other parts of the world contacted us and asked to participate. We added events in Australia, Uzbekistan and Nepal. For each of these locations, we expected small numbers. In Australia, we had "Team April"; she ran a self-organized 1/2 marathon from Bong Bong to Burradoo. In Uzbekistan, we had "Team Singer", a family of four who organized a weekend of events, which started with a tour of Tashkent and ended with a hike on Sunday to 1,000-year-old petroglyphs. In Nepal, we also thought this team would be a bit small. One week before the event, I only saw two registered participants. Exchanging messages with the team leader, I learned that she had registered 60 children and adults. It was an incredible surprise to learn that 90 registered to raise awareness about RP (I'm tearing-up again as I type this). We cannot thank Kalayani enough for sharing her photos and videos with the group. I think we all agree that it was an amazing start to a beautiful day. In Europe and the UK, friends organized small groups to walk or run or bike together. In the U.S., the Big Ride team biked, camped and biked. All of us connecting via WhatsApp. The on-going posting to the group was truly inspirational. Beyond our Expections When we decided to organize this first fundraiser, we expected between 40-50 friends and family to register. And, thought we would raise between $500 and $1,000. The actual results far exceeded expectations. The first fundraiser had 400 participants in eight countries (Australia, Nepal, Uzbekistan, Germany, Norway, Netherlands, UK and U.S.) and raised in pledged donations $14,000. We're still in a bit of shock with the success. Beyond the larger participation, we're feeling so blessed because of the expressions of love and support. Most donations (nearly 83%) have been received, and we're so appreciative. If you would still like to donate, please make checks payable to "RP Hope" and mail to: If you're in Europe, please email info@rphope.org for details regarding a funds transfer. Since our personal goal is to run this nonprofit with minimal administrative costs, payments via the website are still unavailable. We're waiting for PayPal and Wix to approve RP Hope as a recognized nonprofit which will reduce the online processing fee. (Online processing fees range from 2.9% - 4.5%.) Kindness of Local Support & Our Volunteers We'd like to thank local businesses that allowed us to promote the fundraiser, especially Bagel Alley and ADB Promotions. We couldn't have organized the overall event without the help of family and friends. We'd like to especially thank Mark Jordan, Taylor Nelson, Judi Hagen, Kalayani Bastakoti Adhikari, Schuyler House, Stewart House, Nicole Siemer, April Gough, Christina Delius, Kelly Molloy, Minal Rajan, and Becki Bishop. Your Donation & Medical Research In addition to raising awareness, our mission is to raise fund for treatments that halt or restore sight to those affected by RP. We see promising technologies being developed. For example, in July 2018, the FDA approved the first ever gene therapy to treat patients with the mutation in gene RPE65. Although it's amazing technology, we see two major problems: This technology only works for patients with the RPE65 mutations, which is less than 2% of all RP patients, meaning only 2,000 people will benefit from the therapy. Fortunately, research suggests this technology may be applicable to other gene mutations. Therefore, RP Hope will focus on solutions that bring these genetic therapies to practical application for a broader patient population. The cost of the treatment is extremely high, exceeding $750,000. RP Hope wants to provide a pathway to treatments that is economically feasible. Contest Finalists Although it was near impossible to think of a unique destination after seeing 90 Nepalese children hiking in the Himalaya's, many teams thought of creative ways to raise awareness about RP. Here are some of the top finalists: Right after the fundraiser, Cate, like all other high school seniors in their spring semester, began exam prep for her AP classes. Just this weekend, she could finally sit and look closely at all of the photos. With careful consideration, the below photo was chosen as the winner of the April 24th Fundraiser Photo Competition. Cate wrote, "this photo illustrates the power of a strong and supportive community, one of the RP Hope's main goals. It was so heartwarming to not only witness the joy of the Sanjiwani School, but also be a part of it. Their encouragement and alacrity during the early hours of the fundraiser was a beaming beginning that uplifted the spirits of the entire event. Thank you once again to everyone who participated and submitted a photo, and to the 'Close Seconds' whose creative photography and planned poses also caught our eye." Final Thank you! Firstly, I would like to preface that there is no amount of appreciation I can show to fully express my gratitude. Every single person who participated in the fundraiser this past Saturday has filled me and my family with so much love and hope. When we began this non-profit, the idea of creating a community and raising funds seemed almost impossible in the time of COVID. But every single person who walked, ran, biked, or swam in the fundraiser showed us the strength of empathy and generosity. Thank you for selflessly dedicating your time and energy to this cause. Thank you for spreading awareness about RP. Thank you for taking risks and joining together in these isolating times to support me and this new non-profit. Thank you for the heartwarming photos and messages that made the event feel all the more momentous and united. Since my diagnosis, I struggled to sustain the ‘glass half full’ mentality, but the compassion and enthusiasm that flowed throughout last Saturday has filled my cup to the brim with optimism and hope. With all of my heart, thank you, and I can’t wait to join you for the next one. - Cate LATE PUBLISHING: Lastly, I wish to offer a sincere apology for the delay in posting this summary. Those in the Netherlands know that I currently supports many organizations. And, planning has been particularly challenging due to COVID restrictions. Once we finalize summer transitions, my focus will be RP Hope.

Al-Khuzaei S, Broadgate S, Foster CR, Shah M, Yu J, Downes SM, Halford S. An Overview of the Genetics of ABCA4 Retinopathies, an Evolving Story. Genes (Basel). 2021 Aug 13;12(8):1241. doi: 10.3390/genes12081241 . PMID: 34440414; PMCID: PMC8392661. Abstract Stargardt disease (STGD1) and ABCA4 retinopathies (ABCA4R) are caused by pathogenic variants in the ABCA4 gene inherited in an autosomal recessive manner. The gene encodes an importer flippase protein that prevents the build-up of vitamin A derivatives that are toxic to the RPE. Diagnosing ABCA4R is complex due to its phenotypic variability and the presence of other inherited retinal dystrophy phenocopies. ABCA4 is a large gene, comprising 50 exons; to date > 2000 variants have been described. These include missense, nonsense, splicing, structural, and deep intronic variants. Missense variants account for the majority of variants in ABCA4. However, in a significant proportion of patients with an ABCA4R phenotype, a second variant in ABCA4 is not identified. This could be due to the presence of yet unknown variants, or hypomorphic alleles being incorrectly classified as benign, or the possibility that the disease is caused by a variant in another gene. This underlines the importance of accurate genetic testing. The pathogenicity of novel variants can be predicted using in silico programs, but these rely on databases that are not ethnically diverse, thus highlighting the need for studies in differing populations. Functional studies in vitro are useful towards assessing protein function but do not directly measure the flippase activity. Obtaining an accurate molecular diagnosis is becoming increasingly more important as targeted therapeutic options become available; these include pharmacological, gene-based, and cell replacement-based therapies. The aim of this review is to provide an update on the current status of genotyping in ABCA4 and the status of the therapeutic approaches being investigated. The aim of this review article is to give an overview of the current status of genotyping in ABCA4 , an update on missing heritability in ABCA4, phenocopies, the effect of genotype on the severity of the phenotype, and assessment techniques to predict the functional consequences of the variants. We will also provide an update on the current therapeutic approaches that are being investigated. Therapies Currently there are no commercially available treatments for ABCA4R/STGD1. Patients are currently advised to avoid supplements containing vitamin A, due to lipofuscin accumulation being seen in Abca4 knockout mice that were given vitamin A [ 163 ]. Wearing protective, dark-tinted glasses in bright conditions is recommended to reduce short wavelength light reaching the retina, thus reducing the risk of light toxicity [ 164 ]. Potential treatments currently being investigated include pharmacological interventions, gene therapy, and stem cell-based therapy approaches (see Table 6 ). Novel therapies are initially investigated in animal models followed by trials in human subjects. Human trials are divided into four phases: Phase I—to assess the safety of the therapy in a small number of subjects; Phase II—to assess efficacy where patients are randomly placed in treatment and placebo arms; Phase III—similarly assesses efficacy but uses a larger cohort of randomized patients; and Phase IV—monitoring the therapy when it becomes available. Pharmacological therapies for ABCA4R are mainly based on targeting aspects of the visual cycle in order to reduce the accumulation of lipofuscin deposits. Table 6 details the effect of the compound and trial results if published. The main advantage of these potential therapies is that they can be taken orally, meaning they are less invasive. Keywords: ABCA4 , Stargardt disease, genetic testing, ABCA4-associated retinopathies, phenocopies READ the entire article

Tal Brandwine, Reut Ifrah, Tzofia Bialistok, Rachel Zaguri, Elisheva Rhodes-Mordov, Liliana Mizrahi-Meissonnier,  Dror Sharon , Vladimir L. Katanaev, Offer Gerlitz, Baruch Minke |  Frontiers in Molecular Neuroscience | 2021 05 July | doi.org/10.3389/fnmol.2021.693967 Dehydrodolichyl diphosphate synthase (DHDDS) is a ubiquitously expressed enzyme that catalyzes cis -prenyl chain elongation to produce the poly-prenyl backbone of dolichol. It appears in all tissues including the nervous system and it is a highly conserved enzyme that can be found in all animal species. Individuals who have biallelic missense mutations in the DHDDS gene are presented with non-syndromic retinitis pigmentosa with unknown underlying mechanism. We have used the Drosophila model to compromise DHDDS ortholog gene ( CG10778 ) in order to look for cellular and molecular mechanisms that, when defective, might be responsible for this retinal disease. The Gal4/UAS system was used to suppress the expression of CG10778 via RNAi-mediated-knockdown in various tissues. The resulting phenotypes were assessed using q-RT-PCR, transmission-electron-microscopy (TEM), electroretinogram, antibody staining and Western blot analysis. Targeted knockdown of CG10778 -mRNA in the early embryo using the actin promoter or in the developing wings using the nub promoter resulted in lethality, or wings loss, respectively. Targeted expression of CG10778 -RNAi using the glass multiple reporter (GMR)-Gal4 driver (GMR-DHDDS-RNAi) in the larva eye disc and pupal retina resulted in a complex phenotype: (a) TEM retinal sections revealed a unique pattern of retinal-degeneration, where photoreceptors R2 and R5 exhibited a nearly normal structure of their signaling-compartment (rhabdomere), but only at the region of the nucleus, while all other photoreceptors showed retinal degeneration at all regions. (b) Western blot analysis revealed a drastic reduction in rhodopsin levels in GMR-DHDDS-RNAi-flies and TEM sections showed an abnormal accumulation of endoplasmic reticulum (ER). To conclude, compromising DHDDS in the developing retina, while allowing formation of the retina, resulted in a unique pattern of retinal degeneration, characterized by a dramatic reduction in rhodopsin protein level and an abnormal accumulation of ER membranes in the photoreceptors cells, thus indicating that DHDDS is essential for normal retinal formation. Introduction The identification of a founder mutation in the dehydrodolichyl diphosphate synthase ( DHDDS ) gene in Ashkenazi Jews (AJ) with non-syndromic retinitis pigmentosa (RP) was reported a decade ago ( Zelinger et al., 2011 ; Züchner et al., 2011 ). A single-nucleotide mutation in the DHDDS gene c.124A > G was found in an AJ family. The p.K42E mutation affects a highly conserved region of the DHDDS protein, which is located in close proximity to a binding site of farnesyl diphosphate. This mutation has subsequently been confirmed in other similar patients and is found in 10–20% of autosomal recessive RP in AJ population ( Zelinger et al., 2011 ). The clinical phenotype of patients who are homozygous for the p.K42E mutation is within the spectrum often described in autosomal recessive RP ( Zelinger et al., 2011 ; Züchner et al., 2011 ). Patients harboring mutations in DHDDS demonstrated fundus findings at a relatively early age. Clinically, they demonstrated waxy appearance of the optic nerve head, attenuation of retinal blood vessels and retinal atrophy in the mid and far periphery combined with significant bone spicule-like pigmentation, starting already in their 20s. The atrophic changes spread into the macular area and the pigmentary changes became denser with age ( Kimchi et al., 2018 ). Kinetic visual fields revealed reduced peripheral function in the youngest patients studied and only small central islands of vision remaining later in life. Electroretinogram responses were not detectable in most patients ( Zelinger et al., 2011 ). In the human retina, DHDDS is expressed in the inner segment of photoreceptors, where dolichol biosynthesis is predicted to occur ( Zelinger et al., 2011 ). A possible link between insufficient DHDDS activity and photoreceptor degeneration was investigated in zebrafish, in which the expression of DHDDS was knocked down by morpholino oligonucleotides injected into zebrafish one cell embryos. The results demonstrated that suppression of DHDDS expression in zebrafish led to loss of photoreceptor outer segments and visual function ( Wen et al., 2014 ). The results thus support the hypothesis that insufficient DHDDS function can lead to retinal degeneration. However, an additional study describing a patient with a severe multisystem disease associated with DHDDS deficiency shows that RP is not the only clinical sign in cases of DHDDS deficiency ( Sabry et al., 2016 ). Click here to read the entire article References Kimchi, A., Khateb, S., Wen, R., Guan, Z., Obolensky, A., Beryozkin, A., et al. (2018). Nonsyndromic retinitis pigmentosa in the ashkenazi jewish population: genetic and clinical aspects. Ophthalmology 125, 725–734. doi: 10.1016/j.ophtha.2017.11.014 Sabry, S., Vuillaumier-Barrot, S., Mintet, E., Fasseu, M., Valayannopoulos, V., Héron, D., et al. (2016). A case of fatal Type I congenital disorders of glycosylation (CDG I) associated with low dehydrodolichol diphosphate synthase (DHDDS) activity. Orphanet J. Rare Dis. 11:84. Wen, R., Dallman, J. E., Li, Y., Züchner, S. L., Vance, J. M., Peričak-Vance, M. A., et al. (2014). Knock-down DHDDS expression induces photoreceptor degeneration in zebrafish. Adv. Exp. Med. Biol. 801, 543–550. doi: 10.1007/978-1-4614-3209-8_69 Zelinger, L., Banin, E., Obolensky, A., Mizrahi-Meissonnier, L., Beryozkin, A., Bandah-Rozenfeld, D., et al. (2011). A missense mutation in DHDDS, encoding dehydrodolichyl diphosphate synthase, is associated with autosomal-recessive retinitis pigmentosa in ashkenazi jews. Am. J. Hum. Genet. 88, 207–215. doi: 10.1016/j.ajhg.2011.01.002 Züchner, S., Dallman, J., Wen, R., Beecham, G., Naj, A., Farooq, A., et al. (2011). Whole-exome sequencing links a variant in DHDDS to retinitis pigmentosa. Am. J. Hum. Genet. 88, 201–206. doi: 10.1016/j.ajhg.2011.01.001

ProQR news – Positive results achieved in our ongoing Usher syndrome and retinitis pigmentosa research. Stellar study, Phase 1/2 clinical trial showed investigational RNA therapy QR-421a is effective and safe. ProQR plans to start final phase trials for people with USH2A mediated retinitis pigmentosa. We are pleased to announce the positive results of our Usher syndrome and retinitis pigmentosa clinical study Stellar, which has met all its stated objectives. This marks a crucial milestone in our ongoing inherited retinal disease research. Read more.

Wei Chiu, Ting-Yi Lin, Yun-Chia Chang, Henkie Isahwan-Ahmad Mulyadi Lai, Shen-Che Lin, Chun Ma, Aliaksandr A. Yarmishyn, Shiuan-Chen Lin, Kao-Jung Chang, Yu-Bai Chou, Chih-Chien Hsu, Tai-Chi Lin, Shih-Jen Chen, Yueh Chien, Yi-Ping Yang, and De-Kuang Hwang Abstract: Inherited retinal dystrophies (IRDs) are a group of rare eye diseases caused by gene muta- tions that result in the degradation of cone and rod photoreceptors or the retinal pigment epithelium. Retinal degradation progress is often irreversible, with clinical manifestations including color or night blindness, peripheral visual defects and subsequent vision loss. Thus, gene therapies that restore functional retinal proteins by either replenishing unmutated genes or truncating mutated genes are needed. Coincidentally, the eye’s accessibility and immune-privileged status along with major advances in gene identification and gene delivery systems heralded gene therapies for IRDs. Among these clinical trials, voretigene neparvovec-rzyl (Luxturna), an adeno-associated virus vector-based gene therapy drug, was approved by the FDA for treating patients with confirmed biallelic RPE65 mutation-associated Leber Congenital Amaurosis (LCA) in 2017. This review includes current IRD gene therapy clinical trials and further summarizes preclinical studies and therapeutic strategies for LCA, including adeno-associated virus-based gene augmentation therapy, 11-cis-retinal replacement, RNA-based antisense oligonucleotide therapy and CRISPR-Cas9 gene-editing therapy. Understand- ing the gene therapy development for LCA may accelerate and predict the potential hurdles of future therapeutics translation. It may also serve as the template for the research and development of treatment for other IRDs. To read more, download the journal article.

The FDA has granted orphan drug designation for chemically induced photoreceptor-like cells to treat retinitis pigmentosa (RP), according to a press release from CiRC Biosciences. The technology “enables direct chemical transdifferentiation of fibroblasts into other cell types using a cocktail of small molecules in a chemical conversion process that takes less than 2 weeks,” the release said. Click here to read the rest of the article. Printed in Ocular Surgery News | March 15, 2021

Downes SM, Nguyen T, Tai V, Broadgate S, Shah M, Al-Khuzaei S, MacLaren RE, Shanks M, Clouston P, Halford S. | Genes (Basel) | 2020 Dec 12 | 11(12) | page 1497 | doi: 10.3390/genes11121497 Abstract Autosomal recessive retinitis pigmentosa is caused by mutations in over 40 genes, one of which is the ceramide kinase-like gene ( CERKL ). We present a case series of six patients from six unrelated families diagnosed with inherited retinal dystrophies (IRD) and with two variants in CERKL recruited from a multi-ethnic British population. A retrospective review of clinical data in these patients was performed and included colour fundus photography, fundus autofluorescence (AF) imaging, spectral domain–optical coherence tomography (SD–OCT), visual fields and electroretinogram (ERG) assessment where available. Three female and three male patients were included. Age at onset ranged from 7 years old to 45 years, with three presenting in their 20s and two presenting in their 40s. All but one had central visual loss as one of their main presenting symptoms. Four patients had features of retinitis pigmentosa with significant variation in severity and extent of disease, and two patients had no pigment deposition with only macular involvement clinically. Seven variants in CERKL were identified, of which three are novel. The inherited retinopathies associated with the CERKL gene vary in age at presentation and in degree of severity, but generally are characterised by a central visual impairment early on. 1. Introduction Inherited retinal dystrophies (IRD) are a heterogeneous group of disorders associated with the dysfunction or death of photoreceptors, resulting in varying severity of visual loss. IRD has an incidence of 1 in 2000–3000, affecting an estimated two million people worldwide [ 1 ]. Retinitis pigmentosa (RP; Mendelian Inheritance in Man (MIM) #268000) is the most common IRD, affecting approximately 1 in 4000 people [ 2 ], characterised by symptoms of nyctalopia, peripheral visual field loss with a clinical appearance of intraretinal bone spicule pigmentation, pale discs and attenuated vasculature. RP is associated with significant genotypic and phenotypic heterogeneity, with 41 genes described in association with the autosomal recessive form to date (RetNet: https://sph.uth.edu/retnet/accessed 1 July 2020). In 1998, an RP locus (RP26) was mapped to an 11 cM region on chromosome 2q31–32 in a consanguineous Spanish family by Bayes and colleagues [ 3 ]. Subsequently, Tuson and colleagues identified a novel gene from the region that was expressed in the retina, named ceramide kinase-like ( CERKL ) [ 4 ]. The coding exons of CERKL were sequenced in the original RP26 family, and all patients were found to be homozygous for a nonsense mutation c.769C > T, p.Arg257* (now annotated as c.847C > T, p.Arg283*, using the numbering based on transcript NM_001030311 , which consists of 14 exons) [ 4 ]. A further unrelated Spanish family was also identified with the same homozygous change [ 4 ]. CERKL shares 29% identity (50% similarity) with the ceramide kinase (CERK) protein, which phosphorylates ceramide to ceramide 1-phosphate, a sphingolipid metabolite which is involved in proliferation, apoptosis, phagocytosis and inflammation [ 5 ]. As yet, no kinase activity is reported for CERKL and its function remains unclear [ 6 , 7 ]. The CERKL gene is composed of 14 exons but alternative splicing produces multiple transcripts ( Figure 1 ) [ 8 , 9 ]. Most of the previous studies examining variants in CERKL used isoform NM_201548 , but this only contains 13 exons and is missing exon 5. Table 1 lists all the previously published mutations and the novel changes described in the patients in the present study, using NM_001030311 (which consists of 14 coding exons) as the reference. To date, 39 different mutations in CERKL have been identified (see Figure 1 and Table 1 for summary). The majority of these CERKL-associated IRD studies are case reports and many include the results from next-generation sequencing (NGS) testing with minimal phenotype data. There are, however, four studies reporting on at least four families, but these pertain to one ethnicity only in each study (Yemenite Jewish, Spanish, Tunisian and Finnish) [ 10 , 11 , 12 , 13 ]. In these reported populations, CERKL is a significant gene contributing to autosomal recessive RP; presumably due to a founder mutation effect. Click here to read entire article References Berger W., Kloeckener-Gruissem B., Neidhardt J. The molecular basis of human retinal and vitreoretinal diseases. Prog. Retin. Eye Res. 2010;29:335–375. doi: 10.1016/j.preteyeres.2010.03.004. Hartong D.T., Berson E.L., Dryja T.P. Retinitis pigmentosa. Lancet. 2006;368:1795–1809. doi: 10.1016/S0140-6736(06)69740-7. Bayes M., Goldaracena B., Martinez-Mir A., Iragui-Madoz M.I., Solans T., Chivelet P., Bussaglia E., Ramos-Arroyo M.A., Baiget M., Vilageliu L., et al. A new autosomal recessive retinitis pigmentosa locus maps on chromosome 2q31-q33. J. Med. Genet. 1998;35:141–145. doi: 10.1136/jmg.35.2.141. Tuson M., Marfany G., Gonzalez-Duarte R. Mutation of CERKL, a novel human ceramide kinase gene, causes autosomal recessive retinitis pigmentosa (RP26) Am. J. Hum. Genet. 2004;74:128–138. doi: 10.1086/381055. Gomez-Munoz A., Presa N., Gomez-Larrauri A., Rivera I.G., Trueba M., Ordonez M. Control of inflammatory responses by ceramide, sphingosine 1-phosphate and ceramide 1-phosphate. Prog. Lipid Res. 2016;61:51–62. doi: 10.1016/j.plipres.2015.09.002. Bornancin F., Mechtcheriakova D., Stora S., Graf C., Wlachos A., Devay P., Urtz N., Baumruker T., Billich A. Characterization of a ceramide kinase-like protein. Biochim. Biophys. Acta. 2005;1687:31–43. doi: 10.1016/j.bbalip.2004.11.012. Domenech E.B., Andres R., Lopez-Iniesta M.J., Mirra S., Garcia-Arroyo R., Milla S., Sava F., Andilla J., Loza-Alvarez P., de la Villa P., et al. A New Cerkl Mouse Model Generated by CRISPR-Cas9 Shows Progressive Retinal Degeneration and Altered Morphological and Electrophysiological Phenotype. Investig. Ophthalmol. Vis. Sci. 2020;61:14. doi: 10.1167/iovs.61.8.14. Garanto A., Riera M., Pomares E., Permanyer J., de Castro-Miro M., Sava F., Abril J.F., Marfany G., Gonzalez-Duarte R. High transcriptional complexity of the retinitis pigmentosa CERKL gene in human and mouse. Investig. Ophthalmol. Vis. Sci. 2011;52:5202–5214. doi: 10.1167/iovs.10-7101. Tuson M., Garanto A., Gonzalez-Duarte R., Marfany G. Overexpression of CERKL, a gene responsible for retinitis pigmentosa in humans, protects cells from apoptosis induced by oxidative stress. Mol. Vis. 2009;15:168–180. Avela K., Sankila E.M., Seitsonen S., Kuuluvainen L., Barton S., Gillies S., Aittomaki K. A founder mutation in CERKL is a major cause of retinal dystrophy in Finland. Acta Ophthalmol. 2017 doi: 10.1111/aos.13551. Auslender N., Sharon D., Abbasi A.H., Garzozi H.J., Banin E., Ben-Yosef T. A common founder mutation of CERKL underlies autosomal recessive retinal degeneration with early macular involvement among Yemenite Jews. Investig. Ophthalmol. Vis. Sci. 2007;48:5431–5438. doi: 10.1167/iovs.07-0736. Habibi I., Chebil A., Falfoul Y., Allaman-Pillet N., Kort F., Schorderet D.F., El Matri L. Identifying mutations in Tunisian families with retinal dystrophy. Sci. Rep. 2016;6:37455. doi: 10.1038/srep37455. Avila-Fernandez A., Riveiro-Alvarez R., Vallespin E., Wilke R., Tapias I., Cantalapiedra D., Aguirre-Lamban J., Gimenez A., Trujillo-Tiebas M.J., Ayuso C. CERKL mutations and associated phenotypes in seven Spanish families with autosomal recessive retinitis pigmentosa. Investig. Ophthalmol. Vis. Sci. 2008;49:2709–2713. doi: 10.1167/iovs.07-0865.

Published: 27 Nov 2020 |Scientific Reports | Vol 10 | Article: 20770 Shogo Numa, Akio Oishi, Koichiro Higasa, Maho Oishi, Manabu Miyata, Tomoko Hasegawa, Hanako Ohashi Ikeda, Yuki Otsuka, Fumihiko Matsuda, Akitaka Tsujikawa Abstract Next-generation sequencing (NGS) has greatly advanced the studies of causative genes and variants of inherited diseases. While it is sometimes challenging to determine the pathogenicity of identified variants in NGS, the American College of Medical Genetics and Genomics established the guidelines to help the interpretation. However, as to the genetic screenings for patients with retinitis pigmentosa (RP) in Japan, none of the previous studies utilized the guidelines. Considering that EYS is the major causative gene of RP in Japan, we conducted stepwise genetic screening of 220 Japanese patients with RP utilizing the guidelines. Step 1–4 comprised the following, in order: Sanger sequencing for two major EYS founder mutations; targeted sequencing of all coding regions of EYS; whole genome sequencing; Sanger sequencing for Alu element insertion in RP1, a recently determined founder mutation for RP. Among the detected variants, 2, 19, 173, and 1 variant(s) were considered pathogenic and 8, 41, 44, and 5 patients were genetically solved in step 1, 2, 3, and 4, respectively. Totally, 44.5% (98/220) of the patients were genetically solved, and 50 (51.0%) were EYS-associated and 5 (5.1%) were Alu element-associated. Among the unsolved 122 patients, 22 had at least one possible pathogenic variant. Introduction The global estimate of retinitis pigmentosa (RP), the most prevalent form of the inherited retinal dystrophies (IRD) across all nations and ethnicities, is 1:4000, and it is a leading cause of severe visual disabilities and blindness in developed countries. It is clinically and genetically heterogeneous. About 100 causative genes have been identified and novel causative genes and mutations are now being reported annually. Recent studies have revealed the spectrum of causative genes and steadily laid the groundwork for genetic approaches to treatments for IRD. Many clinical trials of gene therapies are also ongoing. The approval of Voretigene neparvovec as the first gene therapy for Leber congenital amaurosis (LCA), signaled the dawn of gene therapy for IRD. Given this background, identifying causative genes and their mutations for each IRD among various ethnicities will become more important. Click here to read entire article Cite this article

Jie Gao, Rehan M Hussain, Christina Y Weng | Clinical Ophthalmology | 13 November  2020 | Vol. 14 | pgs. 3855–3869 |doi:  10.2147/OPTH.S231804 Abstract Subretinal gene therapy trials began with the discovery of RPE65 variants and their association with Leber congenital amaurosis. The RPE65 protein is critical for the normal functioning of the visual phototransduction cascade. RPE65 gene knockout animal models were developed and showed similar diseased phenotypes to their human counterparts. Proof of concept studies were carried out in these animal models using subretinal RPE65 gene replacement therapy, resulting in improvements in various visual function markers including electroretinograms, pupillary light responses, and object avoidance behaviors. Positive results in animal models led to Phase 1 human studies using adeno-associated viral vectors. Results in these initial human studies also showed positive impact on visual function and acceptable safety. A landmark Phase 3 study was then conducted by Spark Therapeutics using a dose of 1.5 x1011 vector genomes after dose-escalation studies confirmed its efficacy and safety. Multi-luminance mobility testing was used to measure the primary efficacy endpoint due to its excellent reliability in detecting the progression of inherited retinal diseases. After the study met its primary endpoint, the Food and Drug Administration approved voretigene neparvovec (Luxturna®) for use in RPE65 -associated inherited retinal diseases. Introduction Inherited retinal diseases (IRDs) are a heterogenous group of disorders characterized by varying degrees of functional vision loss and associated retinal degenerative changes. For the collective 270 gene mutations that have been identified in association with clinically diagnosed IRDs, the incidence is approximately 1 in 2000. 1 , 2 In most IRDs, the visual loss occurs early and can be profound, resulting in significant disability to the patient. The primary site of degeneration usually involves the photoreceptor and retinal pigment epithelium (RPE) complex. IRDs can be classified as either stationary, such as in congenital stationary night blindness (CSNB), or progressive, such as in retinitis pigmentosa (RP). 1 Leber congenital amaurosis (LCA) is one of the most severe types of progressive IRDs, presenting with significant functional vision decline within the first year of life. 3 , 4 This review will provide a brief overview on the RPE65 gene mutation-related dystrophies and focus on the clinical evidence that led to the approval of voretigene neparvovec-rzyl, the first FDA (Food and Drug Administration)-approved gene replacement therapy in the United States and in the European Union. 5 , 6 The RPE65 Gene Gene Function The RPE65 gene encodes for a 65 kDa protein located primarily on the smooth endoplasmic reticulum of RPE cells. 7 Electroretinograms (ERGs) of biallelic knockout ( RPE65 -/-) mice demonstrated diminished or absent waveforms similar to what is seen in humans. Dark-adapted ERGs tended to be worse than light-adapted or flicker ERGs suggesting that rod function is more severely impacted than cone function. In the photoreceptors of these eyes, no detectable rhodopsin was found. In the RPE cells, there was an absence of 11-cis-retinol and an overaccumulation of all-trans-retinol. 8 In initial in vitro studies, human cells were transfected with the RPE65 gene along with a LRAT coenzyme gene, followed by exposure to all-trans-retinol. This resulted in decreased all-trans-retinol levels and a dramatic increase in 11-cis-retinol levels, suggesting that the RPE65 protein plays a direct enzymatic role in the isomerization of 11-trans-retinol to 11-cis-retinol. 9 , 10 Other studies suggest that a non-enzymatic role for the RPE65 protein may exist. One study noted that heterozygous ( RPE65 +/–) mice have a much larger drop in rod pigment recycling than would be expected compared to wild type ( RPE65 +/+) mice if the RPE65 protein was acting solely as an enzyme. This study also showed that when RPE65 -/- mice were given oral 9-cis-retinal supplementation, a small amount of 11-cis-retinal could be detected. The author suggested that other proteins may be involved in the visual cycle pathway that eventually produces 11-cis-retinal. They concluded that the RPE65 protein could either have a dual function as both an isomerase enzyme and a structural protein or as an organizer protein involved in the distribution of retinyl esters within the RPE cells. 11 Currently, the exact function of the RPE65 protein is still debated, but what is known is that 11-cis-retinol is converted to 11-cis-retinal which is required by the photoreceptor outer segments in order to combine with opsin and produce the visual pigment that is responsible for detecting light and initiating the phototransduction cascade. 7 Click here to read entire article References Kutluer M, Huang L, Marigo V. Targeting molecular pathways for the treatment of inherited retinal degeneration. Neural Regen Res . 2020;15(10):1784–1791. doi: 10.4103/1673-5374.280303 RetNet. Summaries of genes and loci causing retinal diseases. 2-14-2020. [cited May 5, 2020] Available from: https://sph.uth.edu/retnet/sum-dis.htm . Accessed October23, 2020. Hanein S, Perrault I, Gerber S, et al. Leber congenital amaurosis: comprehensive survey of the genetic heterogeneity, refinement of the clinical definition, and genotype-phenotype correlations as a strategy for molecular diagnosis. Hum Mutat . 2004;23(4):306–317. doi: 10.1002/humu.20010 Gu S-M, Thompson DA, Srikumari CR, et al. Mutations in RPE65 cause autosomal recessive childhood–onset severe retinal dystrophy. Nat Genet . 1997;17(2):194–197. doi: 10.1038/ng1097-194 Novartis announces landmark EU approval for one-time gene therapy Luxturna® to restore vision in people with rare inherited retinal disease. November 23, 2018. [cited May 5, 2020] Available from: https://novartis.gcs-web.com/Novartis-announces-landmark-EU-approval-for-one-time-gene-therapy-Luxturna-to-restore-vision-in-people-with-rare-inherited-retinal-disease . Accessed October23, 2020. LUXTURNA (voretigene neparvovec-rzyl) US full prescribing information. 2017. [cited May 5, 2020] Available from: https://sparktx.com/LUXTURNA_US_Prescribing_Information.pdf . Accessed October23, 2020. Thompson DA, Gal A. Vitamin A metabolism in the retinal pigment epithelium: genes, mutations, and diseases. Prog Retin Eye Res . 2003;22(5):683–703. doi: 10.1016/S1350-9462(03)00051-X Redmond TM, Yu S, Lee E, et al. Rpe65 is necessary for production of 11-cis-vitamin A in the retinal visual cycle. Nat Genet . 1998;20(4):344–351. doi: 10.1038/3813 Redmond TM, Poliakov E, Yu S, Tsai JY, Lu Z, Gentleman S. Mutation of key residues of RPE65 abolishes its enzymatic role as isomerohydrolase in the visual cycle. Proc Natl Acad Sci USA . 2005;102(38):13658–13663. doi: 10.1073/pnas.0504167102 Moiseyev G, Chen Y, Takahashi Y, Wu BX, Ma JX. RPE65 is the isomerohydrolase in the retinoid visual cycle. Proc Natl Acad Sci USA . 2005;102(35):12413–12418. doi: 10.1073/pnas.0503460102 Van Hooser JP, Aleman TS, He YG, et al. Rapid restoration of visual pigment and function with oral retinoid in a mouse model of childhood blindness. Proc Natl Acad Sci USA . 2000;97(15):8623–8628. doi: 10.1073/pnas.150236297

University of California Television (UCTV) "The story of how CIRM-funded research helped preserve vision for Rosie Barrero, who was diagnosed with Retinitis Pigmentosa as a child, and faced the prospect of losing her vision as she became a new mother." Source: https://youtu.be/BjcnCD4kkBo

Avigail Beryozkin , Samer Khateb , Carlos Alberto Idrobo-Robalino , Muhammad Imran Khan , Frans P. M. Cremers , Alexey Obolensky , Mor Hanany , Eedy Mezer , Itay Chowers , Hadas Newman , Tamar Ben-Yosef , Dror Sharon , Eyal Banin  | Scientific Reports | 10 | 16 September 2020 | https://doi.org/10.1038/s41598-020-72028-0 Abstract FAM161A mutations are the most common cause of autosomal recessive retinitis pigmentosa in the Israeli-Jewish population. We aimed to characterize the spectrum of FAM161A -associated phenotypes and identify characteristic clinical features. We identified 114 bi-allelic FAM161A patients and obtained clinical records of 100 of these patients. The most frequent initial symptom was night blindness. Best-corrected visual acuity was largely preserved through the first three decades of life and severely deteriorated during the 4th–5th decades. Most patients manifest moderate-high myopia. Visual fields were markedly constricted from early ages, but maintained for decades. Bone spicule-like pigmentary changes appeared relatively late, accompanied by nummular pigmentation. Full-field electroretinography responses were usually non-detectable at first testing. Fundus autofluorescence showed a hyper-autofluorescent ring around the fovea in all patients already at young ages. Macular ocular coherence tomography showed relative preservation of the outer nuclear layer and ellipsoid zone in the fovea, and frank cystoid macular changes were very rare. Interestingly, patients with a homozygous nonsense mutation manifest somewhat more severe disease. Our clinical analysis is one of the largest ever reported for RP caused by a single gene allowing identification of characteristic clinical features and may be relevant for future application of novel therapies. Introduction Retinitis pigmentosa [RP (MIM #268000)] is the most prevalent hereditary degeneration of the retina in humans, with a prevalence of 1:4,500 (in Europe and USA) 1 , 2 , 3 , 4 , and 1:2,100 in the vicinity of Jerusalem 5 . RP is genetically and clinically heterogeneous, and is characterized by night blindness, progressive degeneration of photoreceptors leading to gradual loss of peripheral and then central vision, and eventually often leads to blindness 6 . On fundus examination patients typically show waxy pallor of the optic discs, attenuation of retinal vessels, and bone spicule-like pigmentary (BSP) changes, and on functional full field electroretinographic (ERG) testing responses are severely diminished and may be non-detectable 6 , 7 . Genetically, RP can be inherited in all Mendelian modes including autosomal recessive (AR, ~ 30% of patients), autosomal dominant (AD, ~ 20%), X-linked (~ 10%), and the remaining 40% are isolate cases 8 . Currently, mutations in 41 genes were reported to cause non-syndromic ARRP (RetNet, https://sph.uth.edu/retnet/ ), including FAM161A , which has been identified in 2010 simultaneously by others and by us 9 , 10 . FAM161A was found to be localized to the base of the connecting cilium, the basal body region, and the adjacent centriole in photoreceptor cells 11 , 12 , 13 , and is part of the cytoskeleton fraction of the cilia and a component of the human centrosome 14 , 15 . It was also found to be a member of the Golgi-centrosomal interactome, a network of proteins interconnecting Golgi maintenance, intracellular transport and centrosome organization 16 . Since the identification of FAM161A as a cause for ARRP in 2010, 13 pathogenic mutations have been reported (Supplementary Fig. S1 and Supplementary Table S1 ) 9 , 10 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 . Two of these mutations (frameshift c.1355_6del and nonsense c.1567C > T) were originally identified to be relatively common among the Jewish population in Israel 9 and two were reported in Palestinian families 9 , 20 . Mutations in this gene are the most common cause of ARRP in Israel (18.2%, Fig.  1 a) while the frequency elsewhere is relatively low (ranging from 0.003 to 2% 17 , 19 , 21 , 22 , 28 ). To date, a total of 82 patients with ARRP caused by FAM161A mutations have been reported in various publications (42 residing in Israel and 53 worldwide), with a phenotype that largely falls within the spectrum described in other genes causing ARRP 9 , 10 , 17 , 18 , 19 , 20 , 21 , 22 , 28 . In the current study we further explore the clinical phenotype in a large cohort of 100 Israeli patients harboring FAM161A mutations for whom clinical data was available. This aims to provide information on the clinical spectrum and course of disease associated with this gene, including identification of characteristic features. The findings can assist in the evaluation and diagnosis of these patients, provide data on prognosis, and may be relevant for future application of novel therapies such as gene augmentation, including windows for intervention and possible outcome measures. Results Identification of FAM161A mutations We analyzed more than 1,500 index cases from our cohort, who suffer from AR or isolate RP, or other IRD types. Using a number of molecular genetic methods, as detailed above, biallelic FAM161A mutations were identified in 81 index cases, with a total of 114 patients in these 81 families-42 of whom were previously reported 9 , with emphasis on the genetic findings (Supplementary Table S2 ). Click here to read entire article References Rosenberg, T. Epidemiology of hereditary ocular disorders. Dev. Ophthalmol. 37, 16–33 (2003). Bundey, S. & Crews, S. J. A study of retinitis pigmentosa in the City of Birmingham. I Prevalence. J. Med. Genet. 21, 417–420 (1984). Peterlin, B. et al. Prevalence of retinitis pigmentosa in Slovenia. Clin. Genet. 42, 122–123 (1992). Bunker, C. H., Berson, E. L., Bromley, W. C., Hayes, R. P. & Roderick, T. H. Prevalence of retinitis pigmentosa in Maine. Am. J. Ophthalmol. 97, 357–365 (1984). Sharon, D. & Banin, E. Nonsyndromic retinitis pigmentosa is highly prevalent in the Jerusalem region with a high frequency of founder mutations. Mol. Vis. 21, 783–792 (2015). Berson, E. L. Retinitis pigmentosa. The Friedenwald Lecture. Invest. Ophthalmol. Vis. Sci. 34, 1659–1676 (1993). Jacobson, S. G. et al. Normal central retinal function and structure preserved in retinitis pigmentosa. Invest. Ophthalmol. Vis. Sci. 51, 1079–1085. https://doi.org/10.1167/iovs.09-4372 (2010). Hartong, D. T., Berson, E. L. & Dryja, T. P. Retinitis pigmentosa. Lancet 368, 1795–1809. https://doi.org/10.1016/S0140-6736(06)69740-7 (2006). Bandah-Rozenfeld, D. et al. Homozygosity mapping reveals null mutations in FAM161A as a cause of autosomal-recessive retinitis pigmentosa. Am. J. Hum. Genet. 87, 382–391. https://doi.org/10.1016/j.ajhg.2010.07.022 (2010). Langmann, T. et al. Nonsense mutations in FAM161A cause RP28-associated recessive retinitis pigmentosa. Am. J. Hum. Genet. 87, 376–381. https://doi.org/10.1016/j.ajhg.2010.07.018 (2010). Zach, F. et al. The retinitis pigmentosa 28 protein FAM161A is a novel ciliary protein involved in intermolecular protein interaction and microtubule association. Hum. Mol. Genet. 21, 4573–4586. https://doi.org/10.1093/hmg/dds268 (2012). Zach, F. & Stohr, H. FAM161A, a novel centrosomal-ciliary protein implicated in autosomal recessive retinitis pigmentosa. Adv. Exp. Med. Biol. 801, 185–190. https://doi.org/10.1007/978-1-4614-3209-8_24 (2014). Di Gioia, S. A. et al. FAM161A, associated with retinitis pigmentosa, is a component of the cilia-basal body complex and interacts with proteins involved in ciliopathies. Hum. Mol. Genet. 21, 5174–5184. https://doi.org/10.1093/hmg/dds368 (2012). Liu, Q. et al. The proteome of the mouse photoreceptor sensory cilium complex. MCP 6, 1299–1317. https://doi.org/10.1074/mcp.M700054-MCP200 (2007). Jakobsen, L. et al. Novel asymmetrically localizing components of human centrosomes identified by complementary proteomics methods. EMBO J. 30, 1520–1535. https://doi.org/10.1038/emboj.2011.63 (2011). Di Gioia, S. A. et al. Interactome analysis reveals that FAM161A, deficient in recessive retinitis pigmentosa, is a component of the Golgi-centrosomal network. Hum. Mol. Genet. 24, 3359–3371. https://doi.org/10.1093/hmg/ddv085 (2015). Venturini, G. et al. Molecular genetics of FAM161A in North American patients with early-onset retinitis pigmentosa. PLoS ONE 9, e92479. https://doi.org/10.1371/journal.pone.0092479 (2014). Duncan, J. L. et al. Ocular Phenotype of a Family with FAM161A-associated retinal degeneration. Ophthalmic Genet. https://doi.org/10.3109/13816810.2014.929716 (2015).  Van Schil, K. et al. A nonsense mutation in FAM161A is a recurrent founder allele in Dutch and Belgian individuals with autosomal recessive retinitis pigmentosa. Invest. Ophthalmol. Vis. Sci. 56, 7418–7426. https://doi.org/10.1167/iovs.15-17920 (2015). Zobor, D., Balousha, G., Baumann, B. & Wissinger, B. Homozygosity mapping reveals new nonsense mutation in the FAM161A gene causing autosomal recessive retinitis pigmentosa in a Palestinian family. Mol. Vis. 20, 178–182 (2014). Zhou, Y. et al. Whole-exome sequencing reveals a novel frameshift mutation in the FAM161A gene causing autosomal recessive retinitis pigmentosa in the Indian population. J. Hum. Genet. 60, 625–630. https://doi.org/10.1038/jhg.2015.92 (2015). Maranhao, B. et al. Investigating the molecular basis of retinal degeneration in a familial cohort of Pakistani decent by exome sequencing. PLoS ONE 10, e0136561. https://doi.org/10.1371/journal.pone.0136561 (2015). Carss, K. J. et al. Comprehensive rare variant analysis via whole-genome sequencing to determine the molecular pathology of inherited retinal disease. Am. J. Hum. Genet. 100, 75–90. https://doi.org/10.1016/j.ajhg.2016.12.003 (2017). Jespersgaard, C. et al. Molecular genetic analysis using targeted NGS analysis of 677 individuals with retinal dystrophy. Sci. Rep. 9, 1219. https://doi.org/10.1038/s41598-018-38007-2 (2019). Wang, J. et al. Dependable and efficient clinical utility of target capture-based deep sequencing in molecular diagnosis of retinitis pigmentosa. Invest. Ophthalmol. Vis. Sci. 55, 6213–6223. https://doi.org/10.1167/iovs.14-14936 (2014). Glockle, N. et al. Panel-based next generation sequencing as a reliable and efficient technique to detect mutations in unselected patients with retinal dystrophies. EJHG 22, 99–104. https://doi.org/10.1038/ejhg.2013.72 (2014). Ellingford, J. M. et al. Molecular findings from 537 individuals with inherited retinal disease. J. Med. Genet. 53, 761–767. https://doi.org/10.1136/jmedgenet-2016-103837 (2016). Rose, A. M. et al. Diverse clinical phenotypes associated with a nonsense mutation in FAM161A. Eye 29, 1226–1232. https://doi.org/10.1038/eye.2015.93 (2015).

Timothy P Levine | 2020 Aug 27 | Vol 9 | pg. 1052 | doi: 10.12688/f1000research.25870.1. Background FAM161A is a microtubule-associated protein conserved widely across eukaryotes, which is mutated in the inherited blinding disease Retinitis Pigmentosa-28. FAM161A is also a centrosomal protein, being a core component of a complex that forms an internal skeleton of centrioles. Despite these observations about the importance of FAM161A, current techniques used to examine its sequence reveal no homologies to other proteins. Methods Sequence profiles derived from multiple sequence alignments of FAM161A homologues were constructed by PSI-BLAST and HHblits, and then used by the profile-profile search tool HHsearch, implemented online as HHpred, to identify homologues. These in turn were used to create profiles for reverse searches and pair-wise searches. Multiple sequence alignments were also used to identify amino acid usage in functional elements. Results FAM161A has a single homologue: the targeting protein for Xenopus kinesin-like protein-2 (Tpx2), which is a strong hit across more than 200 residues. Tpx2 is also a microtubule-associated protein, and it has been shown previously by a cryo-EM molecular structure to nucleate microtubules through two small elements: an extended loop and a short helix. The homology between FAM161A and Tpx2 includes these elements, as FAM161A has three copies of the loop, and one helix that has many, but not all, properties of the one in Tpx2. Conclusions FAM161A and ­its homologues are predicted to be a previously unknown variant of Tpx2, and hence bind microtubules in the same way. This prediction allows precise, testable molecular models to be made of FAM161A-microtubule complexes. Click here to read entire article