- Genetics of human retinal disease Cone dystrophy (CD) and cone-rod dystrophy (CRD) are heterogeneous diseases in which cone photoreceptor dysfunction or loss precedes/accompanies rod photoreceptor degeneration. Known gene mutations account for less than 25% of the cases. Because of the unique genetic make-up of the Israeli population, genetic studies of rod-dominated dystrophies offer the opportunity to identify novel gene mutations. In collaboration with E. Banin and D. Sharon (Hebrew University, Jerusalem), we selected for whole exome sequencing (WES) three to four individuals and one unaffected control from seven unrelated families with well-documented cone-dominated retinal phenotype. Prior mutation screening had excluded the presence of known causative gene mutations. Three/six families displayed two known missense mutations in the GUCY2D gene (p.R838C and p.R838H), one family a nonsense mutation in CDHR1 gene (p.Q461X) and one family a missense mutation in the C8orf37 gene (p.R177W). Targeted screening of exons 13 and 14 of the GUCY2D gene in 106 additional families led to identification of four more families with mutations in this gene and to the discovery of two highly probable novel disease-causing variants (p.E841K and p.K846N). Our work underscores the value of WES in generating more comprehensive and rapid analyses that can be followed by targeted screens of larger samples to delineate the genetics of retinal disease in unique population cohorts. Leber congenital amaurosis (LCA) encompasses a set of early-onset blinding diseases that are characterized by vision loss, involuntary eye movement, and non-recordable electroretinogram (ERG). At least 20 genes are associated with LCA. The variation in severity of the LCA phenotypes and the presence of more than one potentially disease causing variants in individual patients suggest the involvement of modifier genes in generating clinical differences. We applied whole exome sequencing (WES) to detect a complete spectrum of modifier alleles in LCA caused by RPE65 mutations. After a first set of WES of four members of a LCA family, we identified novel segregating variants in two unlinked genes, RPE65 and CHD7. Secondly, we performed WES on additional 37 LCA patients, including 4 sib-pairs, with known RPE65 mutations. We have identified potential modifiers in novel genes (e.g. CHD7, SETD5, DSCAML1) and in known retinal degeneration genes (ERCC6, FAM161A, NPHP4, USH2A and MYO7A). Our study provides a genome-wide approach for examining rare alleles that may contribute to LCA pathogenesis and should help in elucidating differences in disease manifestation among patients. - Animal models of human retinal disease Mutations in the centrosome-cilia protein 290 kDa (CEP290) gene are a common cause of vision loss in LCA, characterized by early onset visual loss with retention of foveal cone photoreceptors. We have generated several mouse models mutated in Cep290. The hypomorphic Cep290/rd16 mouse model is relatively mild compared to CEP290-LCA in human. On the contrary, most Cep290-/- mice die around weaning from hydrocephalus (see below). A minority of Cep290-/- mice that survive, have a normal lifespan. However, they develop rapid retinal degeneration starting at postnatal day (P)12, with dramatic disruption of outer segment morphogenesis, and progressive kidney pathology. Even more dramatic is the phenotype of the Cep290gt/gt mice that die mid-gestation. Rare Cep290gt/gt mice that are born and survive for 2-3 weeks display hydrocephalus and severely cystic kindneys. The variety of phenotypes we observed in distinct Cep290 mouse alleles are in accordance with the broad spectrum of ciliopathies caused in humans by different CEP290 mutations and can set the stage for developing models for syndromic ciliopathies such as Jourbert and Meckel Gruber syndromes (see also below). As part of a collaboration with several groups at U. Florida, U. Pennsylvania and U. Iowa, we mimicked the human non-syndromic CEP290-LCA disease by introducing the Cep290/rd16 gene mutation in the cone-only Nrl-/- mouse. Structural and functional data collected from rd16;Nrl-/- mice and a cohort of CEP290-LCA patients for comparison revealed remarkable similarities between the natural history of early loss of photoreceptor function with retained cone cell nuclei in CEP290-LCA patients and in the rd16;Nrl-/- murine model. We propose the rd16;Nrl-/- as a model for preclinical testing of therapies for CEP290-LCA, with intervention at P35-40 and follow-up at one month by assaying UV-cone ERG (1). CC2D2A is another centrosome-cilia gene associated with syndromic ciliopathies. In particular, CC2D2A is one of the 10 centrosome-cilia genes associated with Meckel syndrome (MKS). The Cc2d2a-/- mouse that we generated recapitulates features of MKS, including embryonic lethality and multiorgan defects. Mechanistically, lack of nodal cilia could explain the situs inversus phenotype. Furthermore disruption of cilia-based Shh signaling, that is normally necessary to initiate neural tube and limb bud patterning, may account for neural tube defects and polydactyly. At the molecular level, we established the role of Cd2d2a in the formation or stabilization of subdistal appendages, which anchor cytoplasmic microtubules, to initiate the process of cilia biogenesis from the basal body (see EY000450)(4). Mutations in the homeodomain transcription factor CRX lead to an autosomal dominant form of LCA. Upon identification of a spontaneous mouse mutant carrying a dominant frame-shift mutation in Crx (CrxRip), we determined that the mutation results in congenital blindness with no ERG response. Photoreceptor differentiation/maturation is incomplete but no degeneration ensues. Expression of human LCA-associated dominant CRX frame-shift mutations in mouse retina mimicked the CrxRip phenotype and overexpression of wild type Crx in the mutant mice reversed the phenotype. The rod-specific transcription factor NRL was progressively downregulated in CrxRip retina and transgenic expression of NRL partially restored rod differentiation/maturation in CrxRip heterozygous mice. Our data provide insights into the molecular mechanisms underlying CRX mutation-associated congenital blindness involving the transcription factors OTX2 and NRL (see EY000450), and potential therapeutic targets (2). - Mitochondria function in aging and diseased retina We have adapted an in vitro assay to test mitochondrial function measured as oxygen consumption rate in freshly isolated retina punches using the Seahorse, HF24 Analyzer. Our data suggest that mitochondria in young, healthy retina function at maximum capacity and would therefore be most sensitive to minimal homeostatic and/or environmental changes such as those occurring in aging or in pre-disease conditions. - In vitro models of human retinal disease Reprogramming of somatic cells to pluripotency allows de novo differentiation to photorecetors and modeling of retinal disease in a dish using the patients'own cells to study pathogenesis and discover new molecules for therapy. We have generated and are currently characterizing induced pluripotent stem cells (iPSCs) from patients with mutations in CEP290 that present LCA or Joubert syndrome phenotypes. Similarly, we have generated embryonic stem cells (ESCs) and iPSCs from the Cep290/rd16 mouse and controls to complement our human studies with a well characterized animal model (see EY000474). Significance Through the identification of disease-causing or -associated genes, the investigation of gene function and of the molecular mechanisms of disease using animal model and patient-derived iPSCs, we are building the basic scientific knowledge necessary to design targeted and efficacious therapies for retinal degenerative diseases.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIAEY000473-06
Application #
8938331
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
6
Fiscal Year
2014
Total Cost
Indirect Cost
Name
U.S. National Eye Institute
Department
Type
DUNS #
City
State
Country
Zip Code
Roger, Jerome E; Hiriyanna, Avinash; Gotoh, Norimoto et al. (2014) OTX2 loss causes rod differentiation defect in CRX-associated congenital blindness. J Clin Invest 124:631-43
Boye, Shannon E; Huang, Wei-Chieh; Roman, Alejandro J et al. (2014) Natural history of cone disease in the murine model of Leber congenital amaurosis due to CEP290 mutation: determining the timing and expectation of therapy. PLoS One 9:e92928
Veleri, Shobi; Manjunath, Souparnika H; Fariss, Robert N et al. (2014) Ciliopathy-associated gene Cc2d2a promotes assembly of subdistal appendages on the mother centriole during cilia biogenesis. Nat Commun 5:4207
Ma, Wenxin; Cojocaru, Radu; Gotoh, Norimoto et al. (2013) Gene expression changes in aging retinal microglia: relationship to microglial support functions and regulation of activation. Neurobiol Aging 34:2310-21
Li, Linjing; Khan, Naheed; Hurd, Toby et al. (2013) Ablation of the X-linked retinitis pigmentosa 2 (Rp2) gene in mice results in opsin mislocalization and photoreceptor degeneration. Invest Ophthalmol Vis Sci 54:4503-11
Swaroop, Anand (2013) What's in a name? RPGR mutations redefine the genetic and phenotypic landscape in retinal degenerative diseases. Invest Ophthalmol Vis Sci 54:1417
Zahid, Sarwar; Khan, Naheed; Branham, Kari et al. (2013) Phenotypic conservation in patients with X-linked retinitis pigmentosa caused by RPGR mutations. JAMA Ophthalmol 131:1016-25
Branham, Kari; Othman, Mohammad; Brumm, Matthew et al. (2012) Mutations in RPGR and RP2 account for 15% of males with simplex retinal degenerative disease. Invest Ophthalmol Vis Sci 53:8232-7
Priya, Rinki Ratna; Rajasimha, Harsha Karur; Brooks, Matthew J et al. (2012) Exome sequencing: capture and sequencing of all human coding regions for disease gene discovery. Methods Mol Biol 884:335-51
Roger, Jerome E; Ranganath, Keerthi; Zhao, Lian et al. (2012) Preservation of cone photoreceptors after a rapid yet transient degeneration and remodeling in cone-only Nrl-/- mouse retina. J Neurosci 32:528-41

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