PURPOSE. Recessive mutations in CLN7/MFSD8 usually cause variant late-infantile onset neuronal ceroid lipofuscinosis (vLINCL), a poorly understood neurodegenerative condition, though mutations may also cause nonsyndromic maculopathy. A series of 12 patients with nonsyndromic retinopathy due to novel CLN7/MFSD8 mutation combinations were investigated in this study. METHODS. Affected patients and their family members were recruited in ophthalmic clinics at each center where they were examined by retinal imaging and detailed electrophysiology. Whole exome or genome next generation sequencing was performed on genomic DNA from at least one affected family member. Immunofluorescence confocal microscopy of murine retina cross-sections were used to localize the protein. RESULTS. Compound heterozygous alleles were identified in six cases, one of which was always p.Glu336Gln. Such combinations resulted in isolated macular disease. Six further cases were homozygous for the variant p.Met454Thr, identified as a founder mutation of South Asian origin. Those patients had widespread generalized retinal disease, characterized by electroretinography as a rod-cone dystrophy with severe macular involvement. In addition, the photopic single flash electroretinograms demonstrated a reduced b- to a-wave amplitude ratio, suggesting dysfunction occurring after phototransduction. Immunohistology identified MFSD8 in the outer plexiform layer of the retina, a site rich in photoreceptor synapses. CONCLUSIONS. This study highlights a hierarchy of MFSD8 variant severity, predicting three consequences of mutation: (1) nonsyndromic localized maculopathy, (2) nonsyndromic widespread retinopathy, or (3) syndromic neurological disease. The data also shed light on the underlying pathogenesis by implicating the photoreceptor synaptic terminals as the major site of retinal disease.

No description available.

La rétinite pigmentaire est une maladie héréditaire rétinienne menant à la cécité, et pour laquelle il n’existe aucun traitement. La cause la plus fréquente des formes autosomiques dominantes de la maladie est une mutation ponctuelle dans le gène de la rhodopsine (RHO) induisant la mort des photorécepteurs (PR). Pour éviter la dégénérescence des PR, la stratégie thérapeutique doit supprimer l’expression de la protéine mutante tout en restaurant celle de la protéine normale et ce à un niveau physiologique. Mon projet était de réparer le pré-ARNm RHO par la technologie SMaRT (trans-épissage (TE) médié par le splicéosome). Ceci nécessite d’introduire par transfert de gène, dans la cellule cible, un ARN exogène, appelé PTM pour molécule pre-ARNm de TE, pouvant induire un épissage en trans.Nous avons créé 20 PTM différents et obtenu un taux maximal de TE in vitro de 40% après co-transfection transitoire des constructions RHO et PTM dans les cellules HEK293T. Nous avons générés des lignées cellulaires d’expression stable de RHO normale ou mutée par transduction lentivirale. Alors que la RHO normale se localise à la membrane plasmique, la mutation induit la rétention cytoplasmique de la protéine. La transfection du PTM dans la lignée cellulaire de RHO mutée a induit du TE, capable de restaurer partiellement la localisation de la RHO réparée à la membrane.Nous avons alors testé le TE in vivo dans un modèle murin humanisé de rétinite pigmentaire. L’injection sous-rétinienne d’un AAV2/8-bRho-PTM a permis le TE in vivo, mais n’a pas suffi à prévenir la dégénérescence des PR observée par SD-OCT (technologie que nous avons améliorée au cours de ce projet). / Retinitis pigmentosa is an hereditary retinal dystrophy involving degeneration of photoreceptors leading to blindness and for which there is currently no treatment. The most frequent cause of autosomal dominant forms of the disease is a point mutation in the rhodopsin gene (RHO). Therapeutic strategy should both suppress mutant protein expression and restore that of the normal one to physiologic level to prevent photoreceptor degeneration. My PhD project was to repair RHO pre-mRNA by SMaRT (Spliceosome Mediated RNA Trans-splicing) technology. This implies to introduce by gene transfer into the target cell an exogenous RNA, called PTM for Pre-mRNA Trans-splicing Molecule. This one was able to promote a splice reaction in trans, leading to the replacement of the mutated exons. We designed 20 different PTM and obtained in vitro a maximum trans-splicing rate of 40% after transient co-transfection of PTM and RHO constructs in HEK293T cells. We then created WT or mutated RHO stable expression cell lines by lentiviral transduction. Mutation induced retention of the protein into the cytoplasm, while the WT RHO was localized to the plasma membrane. We observed that the PTM transfection in the mutated RHO cell line induced trans-splicing, which was able to partially restore localization to the plasma membrane of repaired RHO. We then tested trans-splicing in vivo in mRho+/- RHO P347S+ mice, a humanized heterozygous mouse model of retinitis pigmentosa. After subretinal injection of AAV2/8-bRho-PTM we observed that trans-splicing occurred in vivo. Unfortunately we did not observe by SD-OCT (a technology that we improve in this project) any rescue of the degenerative phenotype.

京都大学 / 0048 / 新制・課程博士 / 博士(社会健康医学) / 甲第20057号 / 社医博第75号 / 新制||社医||9(附属図書館) / 京都大学大学院医学研究科社会健康医学系専攻 / (主査)教授 山田 亮, 教授 小泉 昭夫, 教授 松田 文彦 / 学位規則第4条第1項該当 / Doctor of Public Health / Kyoto University / DFAM

Yes / Fundus-controlled perimetry (FCP, also called 'microperimetry') allows for spatially-resolved mapping of visual sensitivity and measurement of fixation stability, both in clinical practice as well as research. The accurate spatial characterization of visual function enabled by FCP can provide insightful information about disease severity and progression not reflected by best-corrected visual acuity in a large range of disorders. This is especially important for monitoring of retinal diseases that initially spare the central retina in earlier disease stages. Improved intra- and inter-session retest-variability through fundus-tracking and precise point-wise follow-up examinations even in patients with unstable fixation represent key advantages of these technique. The design of disease-specific test patterns and protocols reduces the burden of extensive and time-consuming FCP testing, permitting a more meaningful and focused application. Recent developments also allow for photoreceptor-specific testing through implementation of dark-adapted chromatic and photopic testing. A detailed understanding of the variety of available devices and test settings is a key prerequisite for the design and optimization of FCP protocols in future natural history studies and clinical trials. Accordingly, this review describes the theoretical and technical background of FCP, its prior application in clinical and research settings, data that qualify the application of FCP as an outcome measure in clinical trials as well as ongoing and future developments.

Introdução: A Retinose Pigmentar (RP) é uma doença hereditária da retina, caracterizada por perda da função visual, principalmente devido à degeneração dos fotorreceptores (bastonetes e cones). Objetivo: Avaliar os efeitos de uma única injeção intravítrea de fração mononuclear de células da medula óssea (FMMO) CD34+ em pacientes portadores de RP. Métodos: Ensaio clínico aberto, não randomizado, prospectivo, observador mascarado, no qual 20 pacientes, portadores de RP, com boa fixação ao exame de campo visual, foram incluídos. Única injeção intravítrea (IIV) de FMMO foi aplicada em apenas um dos olhos de cada paciente, enquanto que os olhos contralaterais serviram como controle e foram submetidos à injeção simulada. As avaliações incluíram: melhor acuidade visual corrigida (MAVC); campo visual estático - estratégia 30-2 (Octopus 900); microperimetria (MAIA - Center Vue) para avaliar estabilidade de fixação e sensibilidade macular; eletrorretinografia de campo total (ERG) e multifocal (mfERG) - padrão da ISCEV usando aparelho Espion E2 (Diagnosys LLC) e tomografia de coerência óptica (OCT). Os exames foram realizados antes da injeção e 4, 16, 32 e 48 semanas após. Resultados: Não houve diferença significativa na MAVC durante o seguimento. A diferença entre MAVC medida após 48 semanas e a basal foi de -0,04 ? 0,02 logMAR nos olhos tratados frente a -0,03 ? 0,01 logMAR nos controles (p=0,3898). A melhora da sensibilidade macular foi discretamente maior nos olhos com FMMO: 1,0 ? 0,5 dB do que nos olhos contralaterais: 0,2 ? 0,5 dB, mas sem significância estatística (p=0,0569). Não se observou mudança na estabilidade de fixação. A perda de desvio médio (MD) do campo visual dos olhos tratados (0,33 ? 0,70 dB) foi discretamente menor do que nos olhos controle (1,12 ? 0,58 dB) (p=0,0761). Nenhuma diferença significativa foi observada nas amplitudes e latências das respostas eletrorretinográficas durante o período avaliado. Não se verificou nenhuma complicação e nem efeito colateral após a injeção. Conclusão: A aplicação intravítrea de FMMO contendo células CD34+ mostrou-se segura em pacientes com RP. Observou-se, ainda, discreta melhora na sensibilidade macular, mas esta não foi significativa estatisticamente. Estudos futuros são necessários para esclarecer o potencial uso dessas células em distrofias retinianas. / Introduction: Retinitis pigmentosa (RP) is a hereditary disease of the retina, characterized by loss of visual function, mainly due to degeneration of the photoreceptors (rods and cones). Objective: To evaluate the effects of a single intravitreal injection of bone marrow mononuclear fraction (BMMF) containing CD34+ cells in patients with RP. Methods: Open trial, non-randomized, prospective, masked observer, in which 20 patients with RP with good fixation in visual field examination were included. Single intravitreal injection of BMMF was performed in only one eye of each patient, while the contralateral eyes served as control and underwent shaw injection. Evaluations included: best corrected visual acuity (BCVA); static visual field - strategy 30-2 (Octopus 900); microperimetry (MAIA - Center Vue) to evaluate fixation stability and macular sensitivity; full-field (ERG) and multifocal (mfERG) electroretinograms according to the ISCEV using Espion E2 (Diagnosys LLC) and optical coherence tomography (OCT). The exams were performed before the injection and 4, 16, 32 and 48 weeks after. Results: There was no significant difference in BCVA during follow-up. The difference measured in BCVA between 48 weeks and baseline was 0.04 ? 0.02 logMAR in treated eyes versus -0.03 ? 0.01 logMAR in controls (p=0.3898). The improvement in macular sensitivity was slightly higher in BMMF eyes: 1.0 ? 0.5 dB than in contralateral eyes: 0.2 ? 0.5 dB, but without statistical significance (p=0.0569). No change in fixation stability was observed. The mean deviation loss (MD) of the visual field in treated eyes (0.33 ? 0.70 dB) was slightly lower than in the control eyes (1.12 ? 0.58 dB) (p=0.0761). No significant difference was observed evaluating amplitudes and latencies of ERG and mfERG responses during the follow-up. No complications or side effects were observed after the injection. Conclusion: The intravitreal injection of BMMF containing CD34 + cells was shown to be safe in patients with RP. There was still a slight improvement in macular sensitivity, but this was not statistically significant. Future studies are needed to clarify the potential use of these cells in retinal dystrophies.

no / Retinal dystrophies are an overlapping group of genetically heterogeneous conditions resulting from mutations in more than 250 genes. Here we describe five families affected by an adult-onset retinal dystrophy with early macular involvement and associated central visual loss in the third or fourth decade of life. Affected individuals were found to harbor disease-causing variants in DRAM2 (DNA-damage regulated autophagy modulator protein 2). Homozygosity mapping and exome sequencing in a large, consanguineous British family of Pakistani origin revealed a homozygous frameshift variant (c.140delG [p.Gly47Valfs∗3]) in nine affected family members. Sanger sequencing of DRAM2 in 322 unrelated probands with retinal dystrophy revealed one European subject with compound heterozygous DRAM2 changes (c.494G>A [p.Trp165∗] and c.131G>A [p.Ser44Asn]). Inspection of previously generated exome sequencing data in unsolved retinal dystrophy cases identified a homozygous variant in an individual of Indian origin (c.64_66del [p.Ala22del]). Independently, a gene-based case-control association study was conducted via an exome sequencing dataset of 18 phenotypically similar case subjects and 1,917 control subjects. Using a recessive model and a binomial test for rare, presumed biallelic, variants, we found DRAM2 to be the most statistically enriched gene; one subject was a homozygote (c.362A>T [p.His121Leu]) and another a compound heterozygote (c.79T>C [p.Tyr27His] and c.217_225del [p.Val73_Tyr75del]). DRAM2 encodes a transmembrane lysosomal protein thought to play a role in the initiation of autophagy. Immunohistochemical analysis showed DRAM2 localization to photoreceptor inner segments and to the apical surface of retinal pigment epithelial cells where it might be involved in the process of photoreceptor renewal and recycling to preserve visual function.

No description available.