Retinal degeneration is the progressive loss of neurons lining the posterior surface of the eye. Loss of a certain group of neurons called rod photoreceptors can occur as the result of genetic mutation. In humans, and in mammalian models of retinal degeneration, the death of these cells is permanent, and often followed by cone photoreceptor death, which leads to blindness. As a step towards understanding the implications of rod cell death in the retina, we generated transgenic X. laevis that expressed a novel form of caspase-9, with binding domains specific to the compound AP20187. We treated these transgenic animals with AP20187 and caused rod cell death by apoptosis in tadpoles and post metamorphic animals. Peak rod apoptosis occurred two days after drug exposure. We adapted an electroretinography apparatus, and protocols designed for mammals to measure functional changes in X. laevis rod and cone derived responses. We observed delayed secondary cone cell dysfunction after induced rod cell apoptosis, which was subsequently restored. These animals provide a simple and clinically relevant model of diseases like Retinitis pigmentosa, in which we will be able to probe in detail the mechanisms that govern cone cell dysfunction as a consequence of rod apoptosis. The unique ability of this species to recover from this insult will provide clues towards initiating similar recovery in humans.

Retinal degeneration is the progressive loss of neurons lining the posterior surface of the eye. Loss of a certain group of neurons called rod photoreceptors can occur as the result of genetic mutation. In humans, and in mammalian models of retinal degeneration, the death of these cells is permanent, and often followed by cone photoreceptor death, which leads to blindness. As a step towards understanding the implications of rod cell death in the retina, we generated transgenic X. laevis that expressed a novel form of caspase-9, with binding domains specific to the compound AP20187. We treated these transgenic animals with AP20187 and caused rod cell death by apoptosis in tadpoles and post metamorphic animals. Peak rod apoptosis occurred two days after drug exposure. We adapted an electroretinography apparatus, and protocols designed for mammals to measure functional changes in X. laevis rod and cone derived responses. We observed delayed secondary cone cell dysfunction after induced rod cell apoptosis, which was subsequently restored. These animals provide a simple and clinically relevant model of diseases like Retinitis pigmentosa, in which we will be able to probe in detail the mechanisms that govern cone cell dysfunction as a consequence of rod apoptosis. The unique ability of this species to recover from this insult will provide clues towards initiating similar recovery in humans.

Photoreceptor degeneration due to retinitis pigmentosa (RP) is a primary cause of inherited retinal blindness. Photoreceptor cell-replacement therapies may hold the potential for repair in a degenerate retina, by reinstating light sensitive cells to project and form connections with downstream retinal cells and finally the visual cortex. Patient-specific induced pluripotent stem cells (iPSc) could provide an autologous source of cells to replace lost tissue. However, the use of patient-derived iPSc would require that the disease-causing gene mutation be corrected in cells before transplantation. Ex vivo gene therapy of mouse photoreceptor precursor (PhRP) cells and subretinal transplantation of treated cells are here studied in a disease-specific animal model of RP; rhodopsin was ectopically expressed ex vivo in rod precursor cells, sourced from a transgenic model lacking the rhodopsin gene. Treated rod precursors were here transplanted in mice of the same disease model and are shown to gain expression of rhodopsin and mature to regenerate the absent outer nuclear layer (ONL) of degenerate mice. Visual function, assayed in the same animals before and after transplantation, was restored in animals which had no rod function at baseline. Delivery of the rhodopsin gene by both an adeno associated viral (AAV) vector and a non-viral minicircle DNA vector developed here for ex vivo gene delivery to rod photoreceptor precursors showed comparable efficiency and sustained expression. The non-viral minicircle method provides a novel system for efficient photoreceptor therapy and may offer a platform of genetic treatment of photoreceptor degenerations in which the gene in focus exceeds the size limit for packaging in AAV. Human embryonic stem cell (ESC) and human iPSC-derived PhRPs were also transplanted in mice with complete ONL degeneration and were able to reform the lost photoreceptor layer and mature in the host retina. Human cells developed light-sensitive outer segments, and reconnect with host neurons downstream to improve vision in previously blind mice. Efficient transplantation of ex vivo genetically treated rod precursors and human stem cell-derived PhRPs in animal models of progressive RP may provide a clinically-relevant model for the investigation of cell-replacement therapy for photoreceptor regeneration in retinal disease.

Retinal degeneration is the progressive loss of neurons lining the posterior surface of the eye. Loss of a certain group of neurons called rod photoreceptors can occur as the result of genetic mutation. In humans, and in mammalian models of retinal degeneration, the death of these cells is permanent, and often followed by cone photoreceptor death, which leads to blindness. As a step towards understanding the implications of rod cell death in the retina, we generated transgenic X. laevis that expressed a novel form of caspase-9, with binding domains specific to the compound AP20187. We treated these transgenic animals with AP20187 and caused rod cell death by apoptosis in tadpoles and post metamorphic animals. Peak rod apoptosis occurred two days after drug exposure. We adapted an electroretinography apparatus, and protocols designed for mammals to measure functional changes in X. laevis rod and cone derived responses. We observed delayed secondary cone cell dysfunction after induced rod cell apoptosis, which was subsequently restored. These animals provide a simple and clinically relevant model of diseases like Retinitis pigmentosa, in which we will be able to probe in detail the mechanisms that govern cone cell dysfunction as a consequence of rod apoptosis. The unique ability of this species to recover from this insult will provide clues towards initiating similar recovery in humans. / Medicine, Faculty of / Graduate

Final publication is available at http://dx.doi.org/10.1016/j.exer.2012.08.013. Congrong Guo, Atsushi Otani, Akio Oishi, Hiroshi Kojima, Yukiko Makiyama, Satoko Nakagawa, Nagahisa Yoshimura, Knockout of ccr2 alleviates photoreceptor cell death in a model of retinitis pigmentosa, Experimental Eye Research, Volume 104, November 2012, Pages 39-47, ISSN 0014-4835 / 京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18680号 / 医博第3952号 / 新制||医||1007(附属図書館) / 31613 / 京都大学大学院医学研究科医学専攻 / (主査)教授 長澤 丘司, 教授 伊藤 壽一, 教授 長田 重一 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

This document only includes an excerpt of the corresponding thesis or dissertation. To request a digital scan of the full text, please contact the Ruth Lilly Medical Library's Interlibrary Loan Department (rlmlill@iu.edu).

by Zhang Chun. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (p. 154-197). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstract in Chinese and English.

Inherited retinal dystrophies encompass a broad group of genetic disorders affecting visual functions in as high as 1 in 3,000 individuals around the world. Common symptoms include loss of central, periphery, or night visions, and in severe cases progression to complete blindness. Syndromic forms also exist involving abnormalities in other parts of the body. Currently, more than 250 genes representing a wide variety of functional roles have been shown to be responsible for the disease phenotypes. Moreover, mutations in the same gene sometimes cause different phenotypes while mutations in multiple genes can give rise to the same clinical subtype, further demonstrating the level of complexity in these disorders. Such genetic heterogeneity has substantially complicated the process of pinpointing precise genetic causes underlying these conditions. The goal of my thesis research is to clarify the genetic causes underlying retinal dystrophies, with a primary focus on phenotypes resembling ABCA4-associated diseases and retinitis pigmentosa in both syndromic and non-syndromic forms. Recent advances in the next-generation sequencing (NGS), the high-throughput, ‘deep’ sequencing technology, have enabled several novel genes to be identified, or found new mutations in known genes. Nevertheless, a substantial fraction of unsolved cases still remain. The primary work in this thesis involves utilizing NGS, particularly whole-exome sequencing, to identify disease-causal mutations in families where at least one parent and affected or unaffected siblings are available. Determining all genetic variation underlying retinal diseases is necessary for precise molecular genetic diagnosis and improved prognosis of these conditions. The first part of my thesis highlights the complexity in genetic inheritance of diseases caused by mutations in the ABCA4 gene. In a substantial fraction of Stargardt Disease cases with only one mutation in the ABCA4 coding region, deep sequencing of the entire locus identified the second mutation in the intronic region of the gene in 10% of cases. The genetic heterogeneity of ABCA4 was further demonstrated by the identification of 4 different pathogenic ABCA4 mutations and 4 phenotypes in a single family. These findings epitomized the extremely complex mutational spectrum underlying the ABCA4-associated diseases and suggested thorough sequencing of variations in the entire genomic locus, including copy number variant analysis. In the second part of my thesis, exome-sequencing has led to findings of phenotypic expansions in known disease gene, and in one case the precise molecular diagnosis resulted in an immediate treatment. A family with 2 affected siblings presented novel phenotype of a macular dystrophy caused by mutations in CRB1. In another family where 9 members were affected with late-onset BEM, a mutation was found in CRX given incomplete penetrance. In one family with an affected adult, two well-documented mutations in MMACHC - a gene causal for a potentially debilitating disorder of cobalamin deficiency, were found to segregate with bull’s eye maculopathy (BEM) and minimal systemic features in the proband. Early diagnosis in this patient resulted in hydroxycobalamin treatment for her condition, and possibly an improvement of her systemic prognosis. Together, these findings revealed that clinical phenotype can be very divergent from those described, and only genetic testing can unequivocally determine the cause of a disease. The third part of my thesis work highlights first-time discovery, and co-discovery of new genes associated with retinal diseases. A new form of syndromic RP was investigated in a family presenting a previously undescribed constellation of phenotypic features. Exome sequencing analysis of 3 affected siblings and their unaffected parents revealed deleterious mutations in the RDH11 gene. In another family where 2 affected siblings presented with a remarkably similar phenotype, no mutations in RDH11 were detected. However, analysis of absence of heterozygosity revealed causal mutations in the CWC27 gene. In the search for novel genes in cone-rod dystrophy cases negative of ABCA4 mutations, WES identified new rare, deleterious mutations in RAB28 in two families of Spanish descent. These findings revealed novel genetic causes underlying hereditary retinal diseases, and demonstrated the effectiveness of WES analysis in rare disease gene discovery. In summary, this work represents a comprehensive mutational analysis of inherited retinal dystrophies with complex genotype and phenotype correlations, utilizing next-generation DNA sequencing in large study cohorts. The power of whole-exome sequencing for gene discovery was well demonstrated by unequivocally solving close to 50% of all patients examined in this study. Establishing precise correlations between genotype and clinical phenotype is important for facilitating patient care, counseling, and therapeutic intervention for inherited diseases.

Hundreds of individually rare, but collectively common Mendelian disorders result in visual impairment. One of these disorders is a heterogeneous syndromic form of retinal degeneration, Bardet-Biedl Syndrome (BBS). This disease is an autosomal recessive disorder characterized by retinal degeneration, obesity, learning disabilities, congenital anomalies, and an increased incidence of hypertension and diabetes. Typically, individuals with BBS experience vision loss during childhood leading to blindness by the third decade of life. At least fourteen genes (BBS1-BBS14) are reported to individual cause BBS. This thesis focuses on one of these genes, BBS3, with the overall goal of characterizig the function of BBS3 in terms of both syndromic and non-syndromic retinal degeneration using the zebrafish and mouse model systems. A member of the Ras family of small GTP-binding proteins, BBS3 is postulated to play a role in vesicular transport. A second highly conserved transcript of BBS3, BBS3L, has been identified and is expressed predominantly in the mouse and zebrafish eye. The eye-specific expression of BBS3L facilitates the dissection of BBS function in the retina independent of alterations to other tissues. To this end a Bbs3L knockout mouse was generated and histological analysis at 9 months reveals disorganization of the inner segments, indicative of retinal degeneration. To further evaluate the functional effects of BBS3 deficiency in the eye, an antisense oligonucleotide (Morpholino) approach was utilized to knockdown bbs3 gene expression in zebrafish. Consistent with an eye specific role, knockdown of bbs3L results in mislocalization of the photopigment green cone opsin and reduced visual function, but not abnormalities of the Kupffer's vesicle or delays in intracellular trafficking of melanosomes, both cardinal features of BBS in the zebrafish. To dissect the individual functions of BBS3 and BBS3L, in vitro transcribed wild-type human BBS3 or BBS3L RNA was co-injected with the bbs3 morpholinos. BBS3L RNA, but not BBS3 RNA, restores green opsin localization and vision. Moreover, only BBS3 RNA is sufficient to rescue melanosome transport, a cardinal feature of BBS in the zebrafish. Bbs3L knockout mice as well as a zebrafish bbs3 knockdown model demonstrate that BBS3L is both necessary and sufficient for retinal function and organization. This work was extended to humans by characterizing the A89V missense mutation in BBS3 that results in non-syndromic retinal degeneration. To evaluate the in vivo function of the A89V missense mutation in non-syndromic retinal degeneration and BBS, rescue experiments were performed in the zebrafish. Unlike wild-type BBS3L RNA, BBS3L A89V RNA does not rescue the vision defect seen with loss of bbs3 in zebrafish; however, BBS3 A89V RNA is able to suppress the cardinal zebrafish BBS phenotype of melanosome transport, similar to wild-type BBS3 RNA. These data demonstrate that the BBS3L A89V mutation identified in patients with non-syndromic retinal degeneration is critical and specific for the vision defect.

<p>Detta är en studie om hur det är att studera på Högskolan i Halmstad när man har någon form av funktionsnedsättning. Vi har i denna studie tittat på hur deras funktionsnedsättning har påverkat deras liv allt ifrån när de fick sina diagnoser tills det att de började studera. Vi har även velat få fram hur deras funktionsnedsättning har påverkat deras studier i den bemärkelse att dem har fått stöd och olika hjälpmedel för att klara av dessa. I denna studie så har vi också tagit reda på hur mycket tid och energi som studenterna får lägga ner på sitt skolarbete, men också hur allt detta har påverkat deras sociala tillvaro i och utanför skolan. Vi har även försökt ta fasta på vad studenterna upplever som problematiskt under sin studietid, och tittat på om deras funktionsnedsättning varit en orsakande faktor i detta.</p>