Background Identification of genetic defects and elucidation of underlying pathways of photoreceptor degeneration constitute an essential step in developing targeted therapeutic strategies for retinal neurodegenerative diseases. N-NRL collaborates with numerous clinicians and scientists to identify genes associated with retinitis pigmentosa (RP), Leber congenital amaurosis (LCA), and syndromic diseases involving photoreceptor dysfunction. Genetics of human retinal diseases Retinal diseases often display genetic and clinical variability ascribable to combinations of allelic differences, modifier loci, epigenetic and environmental factors. A modifier gene affects penetrance, expressivity as well as pleiotropy. We are exploring the effect of modifiers in LCA, which often manifests unexplained genetic and clinical variability even in related individuals. Combined with global retinal RNA-Seq and ChIP-Seq data we can apply additional filtering to evaluate candidate genetic variants.
We aim to define a systematic genome-wide approach for searching rare alleles that may contribute to LCA pathogenesis and phenotypic variability. RP defines a large group of inherited retinal disorders, characterized by progressive photoreceptor degeneration and vision loss. A large proportion of RP patients is isolates or simplex, with no known affected family members. RP2 and RPGR genes have been identified as the primary causative genes in X-linked RP (XLRP), as well as in X-linked cone dystrophy (CD) and cone-rod dystrophy (CRD). In collaboration with groups at University of Michigan and University of Pennsylvania, we have identified pathogenic mutations in RPGR and RP2 genes in 15% of simplex male patients screened and proposed RPGR as a first tier gene for screening isolated males with retinal degeneration (1). CD and CRD are clinically and genetically heterogeneous and display various modes of Mendelian inheritance. Mutations in several genes have been identified, however the genetic cause is still unknown for several cases. Upon whole exome sequencing of 27 individuals from eight unrelated Israeli families (in collaboration with Hebrew University), we have identified rare variants in GUCY2D, CDHR1, C8orf37 and CACNA1F, previously associated with CD/CRD. In another autosomal recessive family we have identified a potential causative variant in a gene linked to a syndromic disorder involving loss of vision. We are pursuing exome sequencing of additional Israeli families and investigation of the biological effect(s) of novel gene variants on disease pathology. Animal models of human retinal disease Mutations in RP2 gene are associated with 10-15% of XLRP. In collaboration with colleagues at University of Massachusetts, we investigated the pathogenesis of retinal degeneration in Rp2-/- mice. Despite no overt signs of degeneration, photopic and scotopic ERG declined gradually from one month of age in Rp2-/- retina. Slow, progressive degeneration of photoreceptor membrane discs was associated with mislocalization of cone opsins to the nuclear and synaptic layers, and to reduced rhodopsin content in outer segments (OS), suggesting that RP2 contributes to maintenance of photoreceptor function and that cone opsin mislocalization represents an early step in RP2-associated XLRP (3). Cep290 is a common cause of vision loss in LCA and is involved in several syndromic ciliopathies. Cep290 is essential for photoreceptor ciliogenesis and OS formation. Since the phenotype of the hypomorphic mouse model Cep290rd16 is relatively mild compared to Cep290-associated human pathology, we generated Cep290-/- mice. Most Cep290-/- mice die around weaaning from hydrocephalus. However, tthose that survive have a normal lifespan and develop rapid retinal degeneration starting at postnatal day (P)12. The effects on OS morphogenesis are more pronounced than in Cep290rd16 retinas, supporting a critical role of CEP290 in photoreceptor cilia development. CC2D2A is associated with Meckel (MKS) and Joubert syndrome ciliopathies. The Cc2d2a-/- mouse we generated is embryonic lethal and recapitulates the clinical symptoms of MKS. Embryonic fibroblasts (MEF) from Cc2d2a-/- mice presented defect in cilia basal body in the mother centriole subdistal appendages (SDA), where Cc2d2a is localized. The cilia biogenesis defect was rescued by Cc2d2a transgene. Mutations in transcription factor CRX lead to autosomal dominant LCA. The mechanism of CRX-LCA is not understood. We have identified a mouse with a 1-bp frameshift deletion in Crx coding sequence, similar to many human LCA-causing dominant CRX mutations (CrxRip). Unlike Crx-/- mouse retina, the dominant Crx c.763del1 mutation in CrxRip results in congenital blindness with complete loss of ERG, yet photoreceptors do not degenerate. Dominant CRX frameshift mutations associated with LCA mimic the CrxRip phenotype and can be rescued by Crx. RNA-Seq profiling revealed progressive and complete loss of rod differentiation factor Nrl in CrxRip, while residual Nrl remains in Crx-/- retina. Moreover, Nrl partially restored the rod phenotype in CrxRip/+ mice. Our study provides the mechanism of congenital blindness caused by dominant CRX mutations and should assist in therapeutic design. Altered homeostasis in the aging retina Molecular pathways leading to rod dysfunction in aging are not delineated. By microarray and RNA-Seq profiling of aging mouse rods (flow sorted from Nrl-GFP mice), we have identified changes in gene expression in mitochondrial and proteasomal genes. Although down-regulation of individual genes of the respiratory chain complexes is minimal, the combined effect could be significant in photoreceptors. To test this hypothesis, we have adapted an in vitro assay for measurement of oxygen consumption rate (as an indicator of mitochondrial function) in freshly isolated retina punches using the Seahorse, HF24 Analyzer. The analysis of data from aging and caloric-restricted mice is in progress. To gain additional insights, we have collected human retina, RPE and choroid at different ages and from normal and disease individuals to correlate genotype with aging-associated expression changes using RNA-Seq. Microglia (MG), the resident immune cells of the central nervous system, are thought to contribute to the pathogenesis of age-related neurodegeneration. We collaborated with W. Wong, NEI, to compare gene expression profiles of aging mouse MG. Molecular pathways involving immune function and regulation, angiogenesis, and neurotrophin signaling showed age-related changes. In particular, expression of complement genes, C3 and CFB, previously associated with age-related macular degeneration (AMD), increased with aging, suggesting that senescent MG may contribute to complement dysregulation during disease pathogenesis. The age-related gene expression changes we detected appear to alter MG constitutive support functions and regulation of their activation status (4). 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 disease pathogenesis and discover new molecules for therapy. We are focusing on patients with mutations in CEP290 that present LCA or Joubert syndrome phenotypes. In parallel, we have generated induced pluripotent stem cells (iPSCs) from the Cep290rd16 mouse and controls to complement our human studies using a mouse model of which we have extensive knowledge. Significance We have identified and characterized a few disease genes. Research on animal models has highlighted an interesting convergence on biogenesis and transport functions associated with primary cilia and revealed novel pathways contributing to photoreceptor homeostasis.

National Institute of Health (NIH)
National Eye Institute (NEI)
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
Application #
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
U.S. National Eye Institute
Zip Code
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
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
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
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
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
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
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
Friedman, James S; Ray, Joseph W; Waseem, Naushin et al. (2009) Mutations in a BTB-Kelch protein, KLHL7, cause autosomal-dominant retinitis pigmentosa. Am J Hum Genet 84:792-800