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S P Daiger ,  L S Sullivan ,  S J Bowne | Clinical Genetics | 23 May 2013 | Volume 84, Issue 2 | Pages 132-141 | doi.org/10.1111/cge.12203 Abstract Retinitis pigmentosa (RP) is a heterogeneous set of inherited retinopathies with many disease-causing genes, many known mutations, and highly varied clinical consequences. Progress in finding treatments is dependent on determining the genes and mutations causing these diseases, which includes both gene discovery and mutation screening in affected individuals and families. Despite the complexity, substantial progress has been made in finding RP genes and mutations. Depending on the type of RP, and the technology used, it is possible to detect mutations in 30-80% of cases. One of the most powerful approaches to genetic testing is high-throughput 'deep sequencing', that is, next-generation sequencing (NGS). NGS has identified several novel RP genes but a substantial fraction of previously unsolved cases have mutations in genes that are known causes of retinal disease but not necessarily RP. Apparent discrepancy between the molecular defect and clinical findings may warrant reevaluation of patients and families. In this review, we summarize the current approaches to gene discovery and mutation detection for RP, and indicate pitfalls and unsolved problems. Similar considerations apply to other forms of inherited retinal disease. To purchase the publication, click here

Melissa C. Humbert, Katie Weihbrecht, Charles C. Searby, Yalan Li, Robert M. Pope, Val C. Sheffield, Seongjin Seo | Proceedings of the National Academy of Sciences of the United States of America (PNAS) | 27 Nov 2012 | 109 (48) | 19691-19696 | doi.org/10.1073/pnas.1210916109 Abstract Mutations affecting ciliary components cause a series of related genetic disorders in humans, including nephronophthisis (NPHP), Joubert syndrome (JBTS), Meckel-Gruber syndrome (MKS), and Bardet-Biedl syndrome (BBS), which are collectively termed "ciliopathies." Recent protein-protein interaction studies combined with genetic analyses revealed that ciliopathy-related proteins form several functional networks/modules that build and maintain the primary cilium. However, the precise function of many ciliopathy-related proteins and the mechanisms by which these proteins are targeted to primary cilia are still not well understood. Here, we describe a protein-protein interaction network of inositol polyphosphate-5-phosphatase E (INPP5E), a prenylated protein associated with JBTS, and its ciliary targeting mechanisms. INPP5E is targeted to the primary cilium through a motif near the C terminus and prenyl-binding protein phosphodiesterase 6D (PDE6D) -dependent mechanisms. Ciliary targeting of INPP5E is facilitated by another JBTS protein, ADP-ribosylation factor-like 13B (ARL13B), but not by ARL2 or ARL3. ARL13B missense mutations that cause JBTS in humans disrupt the ARL13B-INPP5E interaction. We further demonstrate interactions of INPP5E with several ciliary and centrosomal proteins, including a recently identified ciliopathy protein centrosomal protein 164 (CEP164). These findings indicate that ARL13B, INPP5E, PDE6D, and CEP164 form a distinct functional network that is involved in JBTS and NPHP but independent of the ones previously defined by NPHP and MKS proteins. Primary cilia are microtubule-based cell surface projections that emanate from the centrosome. This subcellular organelle functions as an antenna, sensing and transducing extracellular signals into the cell, and plays an essential role in regulating multiple cellular processes including the cell cycle, embryonic development, and tissue homeostasis ( 1 – 3 ). Mutations affecting ciliary and centrosomal components underlie a group of related human disorders such as Joubert syndrome (JBTS), Meckel-Gruber syndrome (MKS), nephronophthisis (NPHP), and Bardet-Biedl syndrome (BBS), collectively termed ciliopathies ( 1 – 3 ). Recent protein–protein interaction studies have identified several functional modules or networks involved in these ciliopathies ( 4 ). For example, BBS proteins and intraflagellar transport (IFT) proteins form multiprotein complexes, the BBSome and the IFT complexes, respectively, and these complexes are involved in transporting ciliary proteins. Likewise, NPHP and MKS proteins form a distinct modular complex at the transition zone of primary cilia and regulate ciliary membrane compositions ( 5 – 9 ). However, there are many ciliary and centrosomal proteins [e.g., inositol polyphosphate-5-phosphatase E (INPP5E) and ADP-ribosylation factor-like 13B (ARL13B)] that have not been linked to any of the known functional networks and their precise functions remain to be elucidated. INPP5E encodes an enzyme that hydrolyzes the 5-phosphate of PtdIns(3,4,5)P3 and PtdIns(4,5)P2 and localizes to primary cilia. Mutations in this gene cause JBTS in humans ( 10 , 11 ). In mice, loss of Inpp5e activity results in cystic kidney, bilateral anophthalmia, polydactyly, skeletal defects, cleft palate, and cerebral developmental defects ( 11 ). Inactivation of Inpp5e in adult mice results in obesity and photoreceptor degeneration. Interestingly, many proteins that localize to cilia, including INPP5E, RPGR, PDE6 α and β subunits, GRK1 (Rhodopsin kinase), and GNGT1 (Transducin γ chain), are prenylated (either farnesylated or geranylgeranylated), and mutations in these genes or genes involved in their prenylation (e.g., AIPL1 and RCE1 ) lead to photoreceptor degeneration in vertebrates and humans ( 12 – 18 ). Understanding the molecular mechanisms by which prenylated proteins are transported to primary cilia would have significant ramifications in developing therapeutic strategies to treat blindness and other ciliopathy phenotypes associated with these genes. In this study, we sought to determine the functional network of INPP5E and the mechanisms by which INPP5E is targeted to primary cilia. We determined the ciliary targeting sequence (CTS) of INPP5E, the small GTPase responsible for its ciliary targeting, and an interaction network that connects INPP5E to other known ciliopathy genes and new candidates. To read entire article, click here Reference Goetz SC, Anderson KV. The primary cilium: A signalling centre during vertebrate development. Nat Rev Genet. 2010;11(5):331–344. doi: 10.1038/nrg2774. Singla V, Reiter JF. The primary cilium as the cell’s antenna: Signaling at a sensory organelle. Science. 2006;313(5787):629–633. doi: 10.1126/science.1124534. Mockel A, et al. Retinal dystrophy in Bardet-Biedl syndrome and related syndromic ciliopathies. Prog Retin Eye Res. 2011;30(4):258–274. doi: 10.1016/j.preteyeres.2011.03.001. van Reeuwijk J, Arts HH, Roepman R. Scrutinizing ciliopathies by unraveling ciliary interaction networks. Hum Mol Genet. 2011;20(R2):R149–R157. doi: 10.1093/hmg/ddr354. Williams CL, et al. MKS and NPHP modules cooperate to establish basal body/transition zone membrane associations and ciliary gate function during ciliogenesis. J Cell Biol. 2011;192(6):1023–1041. doi: 10.1083/jcb.201012116. Craige B, et al. CEP290 tethers flagellar transition zone microtubules to the membrane and regulates flagellar protein content. J Cell Biol. 2010;190(5):927–940. doi: 10.1083/jcb.201006105. Garcia-Gonzalo FR, et al. A transition zone complex regulates mammalian ciliogenesis and ciliary membrane composition. Nat Genet. 2011;43(8):776–784. doi: 10.1038/ng.891. Sang L, et al. Mapping the NPHP-JBTS-MKS protein network reveals ciliopathy disease genes and pathways. Cell. 2011;145(4):513–528. doi: 10.1016/j.cell.2011.04.019. Chih B, et al. A ciliopathy complex at the transition zone protects the cilia as a privileged membrane domain. Nat Cell Biol. 2012;14(1):61–72. doi: 10.1038/ncb2410. Bielas SL, et al. Mutations in INPP5E, encoding inositol polyphosphate-5-phosphatase E, link phosphatidyl inositol signaling to the ciliopathies. Nat Genet. 2009;41(9):1032–1036. doi: 10.1038/ng.423. Jacoby M, et al. INPP5E mutations cause primary cilium signaling defects, ciliary instability and ciliopathies in human and mouse. Nat Genet. 2009;41(9):1027–1031. doi: 10.1038/ng.427. Meindl A, et al. A gene (RPGR) with homology to the RCC1 guanine nucleotide exchange factor is mutated in X-linked retinitis pigmentosa (RP3) Nat Genet. 1996;13(1):35–42. doi: 10.1038/ng0596-35. Roepman R, et al. Positional cloning of the gene for X-linked retinitis pigmentosa 3: Homology with the guanine-nucleotide-exchange factor RCC1. Hum Mol Genet. 1996;5(7):1035–1041. doi: 10.1093/hmg/5.7.1035. Huang SH, et al. Autosomal recessive retinitis pigmentosa caused by mutations in the alpha subunit of rod cGMP phosphodiesterase. Nat Genet. 1995;11(4):468–471. doi: 10.1038/ng1295-468. McLaughlin ME, Sandberg MA, Berson EL, Dryja TP. Recessive mutations in the gene encoding the beta-subunit of rod phosphodiesterase in patients with retinitis pigmentosa. Nat Genet. 1993;4(2):130–134. doi: 10.1038/ng0693-130. Yamamoto S, Sippel KC, Berson EL, Dryja TP. Defects in the rhodopsin kinase gene in the Oguchi form of stationary night blindness. Nat Genet. 1997;15(2):175–178. doi: 10.1038/ng0297-175. Sohocki MM, et al. Mutations in a new photoreceptor-pineal gene on 17p cause Leber congenital amaurosis. Nat Genet. 2000;24(1):79–83. doi: 10.1038/71732. Christiansen JR, Kolandaivelu S, Bergo MO, Ramamurthy V. RAS-converting enzyme 1-mediated endoproteolysis is required for trafficking of rod phosphodiesterase 6 to photoreceptor outer segments. Proc Natl Acad Sci USA. 2011;108(21):8862–8866. doi: 10.1073/pnas.1103627108.

Lori S Sullivan ,  Daniel C Koboldt ,  Sara J Bowne ,  Steven Lang ,  Susan H Blanton ,  Elizabeth Cadena ,  Cheryl E Avery , Richard A Lewis ,  Kaylie Webb-Jones ,  Dianna H Wheaton ,  David G Birch ,  Razck Coussa ,  Huanan Ren ,  Irma Lopez , Christina Chakarova ,  Robert K Koenekoop ,  Charles A Garcia ,  Robert S Fulton ,  Richard K Wilson ,   George M Weinstock ,  Stephen P Daig er | Investiga tive Ophthalmology & Visual Science | 2014 Sep 4 | 55 (11) | Pgs. 7147-58 | doi: 10.1167/iovs.14-15419 Abstract Purpose: To identify the cause of retinitis pigmentosa (RP) in UTAD003, a large, six-generation Louisiana family with autosomal dominant retinitis pigmentosa (adRP). Methods: A series of strategies, including candidate gene screening, linkage exclusion, genome-wide linkage mapping, and whole-exome next-generation sequencing, was used to identify a mutation in a novel disease gene on chromosome 10q22.1. Probands from an additional 404 retinal degeneration families were subsequently screened for mutations in this gene. Results: Exome sequencing in UTAD003 led to identification of a single, novel coding variant (c.2539G>A, p.Glu847Lys) in hexokinase 1 (HK1) present in all affected individuals and absent from normal controls. One affected family member carries two copies of the mutation and has an unusually severe form of disease, consistent with homozygosity for this mutation. Screening of additional adRP probands identified four other families (American, Canadian, and Sicilian) with the same mutation and a similar range of phenotypes. The families share a rare 450-kilobase haplotype containing the mutation, suggesting a founder mutation among otherwise unrelated families. Conclusions: We identified an HK1 mutation in five adRP families. Hexokinase 1 catalyzes phosphorylation of glucose to glucose-6-phosphate. HK1 is expressed in retina, with two abundant isoforms expressed at similar levels. The Glu847Lys mutation is located at a highly conserved position in the protein, outside the catalytic domains. We hypothesize that the effect of this mutation is limited to the retina, as no systemic abnormalities in glycolysis were detected. Prevalence of the HK1 mutation in our cohort of RP families is 1%. Introduction Retinitis pigmentosa (RP) is a group of inherited dystrophic disorders of the retina leading to profound loss of vision or blindness. The clinical hallmarks of RP are night blindness, followed by progressive loss of peripheral vision, often culminating in complete blindness. Clinical findings of RP include “bone spicule” pigmentary deposits, retinal vessel attenuation, and characteristic changes in the electroretinograms (ERG). 1   Retinitis pigmentosa affects approximately 1 in 4000 people in the United States, Europe, and Japan. Retinitis pigmentosa is exceptionally heterogeneous with many different genes implicated, many different disease-causing mutations in each gene, and varying clinical presentations even among members of the same family. 2 For nonsyndromic RP, mutations in 24 genes are known to cause autosomal dominant RP (adRP); 45 genes cause recessive RP (arRP), and 3 genes cause X-linked RP (summarized in RetNet, https://sph.uth.edu/retnet/ [in the public domain]) .  The genes found to date as causes of adRP do not fall into a single functional category but include a diverse range of retinal functions, including components of the phototransduction cycle ( RHO , GUCA1A , RDH12 ); pre-mRNA processing factors ( PRPF3 , PRPF4 , PRPF6 , PRPF8 , PRPF31 , SNRNP200 ); structural proteins ( PRPH2 , ROM1 ); ciliary proteins ( RP1 , TOPORS ); transcription factors ( NRL , CRX , NR2E3 ); and a seemingly random assortment of other genes ( BEST1 , CA4 , FSCN2 , IMPDH1 , KLHL7 , RPE65 , SEMA4A ). With current techniques, we can identify likely disease-causing mutations in approximately 75% of patients with adRP (Daiger SP, manuscript in preparation, 2014). While mutations in some known genes may be missed, a number of additional adRP genes remain to be identified. In our University of Texas (UT) adRP cohort, mutations have been found in 205 of 265 families, leaving 60 (23%) with potentially novel adRP genes.  Click here to read entire article References Heckenlively JR. Retinitis Pigmentosa . London: J.B. Lippincott; 1988. Daiger SP. Identifying retinal disease genes: how far have we come, how far do we have to go? Novartis Found Symp . 2004; 255: 17–27, discussion 27–36, 177–178. Sullivan LS Bowne SJ Birch DG Prevalence of disease-causing mutations in families with autosomal dominant retinitis pigmentosa: a screen of known genes in 200 families. Invest Ophthalmol Vis Sci . 2006; 47: 3052–3064. Churchill JD Bowne SJ Sullivan LS Mutations in the X-linked retinitis pigmentosa genes RPGR and RP2 found in 8.5% of families with a provisional diagnosis of autosomal dominant retinitis pigmentosa. Invest Ophthalmol Vis Sci . 2013; 54: 1411–1416. Bowne SJ Sullivan LS Avery CE Mutations in the small nuclear riboprotein 200 kDa gene (SNRNP200) cause 1.6% of autosomal dominant retinitis pigmentosa. Mol Vis . 2013; 19: 2407–2417. Bowne SJ Sullivan LS Mortimer SE Spectrum and frequency of mutations in IMPDH1 associated with autosomal dominant retinitis pigmentosa and Leber congenital amaurosis. Invest Ophthalmol Vis Sci . 2006; 47: 34–42. Sullivan LS Bowne SJ Reeves MJ Prevalence of mutations in eyeGENE probands with a diagnosis of autosomal dominant retinitis pigmentosa. Invest Ophthalmol Vis Sci . 2013; 54: 6255–6261. Sullivan LS Bowne SJ Seaman CR Genomic rearrangements of the PRPF31 gene account for 2.5% of autosomal dominant retinitis pigmentosa. Invest Ophthalmol Vis Sci . 2006; 47: 4579–4588. Gire AI Sullivan LS Bowne SJ The Gly56Arg mutation in NR2E3 accounts for 1-2% of autosomal dominant retinitis pigmentosa. Mol Vis . 2007; 13: 1970–1975. Bowne SJ Sullivan LS Gire AI Mutations in the TOPORS gene cause 1% of autosomal dominant retinitis pigmentosa. Mol Vis . 2008; 14: 922–927. Click here to see all references

Giuseppe Bonapace, Abdul Waheed, Gul N Shah, William S Sly |  Proc National Academy Science  | 2004 Aug 17 | Vol. 101, Issue 33 | 12300-5 | doi: 10.1073/pnas.0404764101 Carbonic anhydrase (CA) IV is a glycosylphosphotidylinositol-anchored enzyme highly expressed on the plasma face of microcapillaries and especially strongly expressed in the choriocapillaris of the human eye. In collaboration with scientists at the University of Cape Town (Rondebosch, South Africa), we recently showed that the R14W mutation in the signal sequence of CA IV, which they identified in patients with the retinitis pigmentosa (RP) 17 form of autosomal dominant RP, results in accumulation of unfolded protein in the endoplasmic reticulum (ER), leading to ER stress, the unfolded protein response, and apoptosis in a large fraction of transfected COS-7 cells expressing mutant, but not wild-type, CA IV. Read more, click here.

Jun Yin, Jan Brocher, Utz Fischer, Christoph Winkler  | Molecular Neurodegeneration | Vol. 6, Issue 56 | 2011 | doi.org/10.1186/1750-1326-6-56 Summary RP mutations have also been identified in a group of housekeeping genes that are involved in pre-mRNA splicing and represent the second-largest contribution to RP after mutations in rhodopsin. These genes include  PRPF3, PRPF8, PRPF31 ,  PAP1  and  SNRN200 . All these genes encode core components of the U4/U6.U5 tri-snRNP complex which constitutes a major building block of the pre-mRNA processing spliceosome. Background Retinitis pigmentosa (RP) is an inherited eye disease characterized by the progressive degeneration of rod photoreceptor cells. Mutations in pre-mRNA splicing factors including PRPF31 have been identified as cause for RP, raising the question how mutations in general factors lead to tissue specific defects. Results We have recently shown that the zebrafish serves as an excellent model allowing the recapitulation of key events of RP. Here we use this model to investigate two pathogenic mutations in PRPF31 , SP117 and AD5, causing the autosomal dominant form of RP. We show that SP117 leads to an unstable protein that is mislocalized to the rod cytoplasm. Importantly, its overexpression does not result in photoreceptor degeneration suggesting haploinsufficiency as the underlying cause in human RP patients carrying SP117. In contrast, overexpression of AD5 results in embryonic lethality, which can be rescued by wild-type Prpf31. Transgenic retina-specific expression of AD5 reveals that stable AD5 protein is initially localized in the nucleus but later found in the cytoplasm concurrent with progressing rod outer segment degeneration and apoptosis. Importantly, we show for the first time in vivo that retinal transcripts are wrongly spliced in adult transgenic retinas expressing AD5 and exhibiting increased apoptosis in rod photoreceptors. Conclusion Our data suggest that distinct mutations in Prpf31 can lead to photoreceptor degeneration through different mechanisms, by haploinsufficiency or dominant-negative effects. Analyzing the AD5 effects in our animal model in vivo , our data imply that aberrant splicing of distinct retinal transcripts contributes to the observed retina defects.

Michael Danciger, Vickie Heilbron, Yong-Qing Gao, Dan-Yun Zhao, Samuel G. Jacobson, and Debora B. Farber | Mol. Vis. | Volume 2 (10) | 1996 |  molvis.org/molvis/v2/a10/ to Based on average estimates of the prevalence of non-syndromic  retinitis pigmentosa  (RP) at 1/4,000, there are approximately 1.5 million people in the world with this inherited, progressive, degenerative disease of the retinal photoreceptor cells which often leads to blindness. About 50% of these cases are inherited in an autosomal recessive manner (AR). With the approach of screening the exons of candidate genes in large numbers of unrelated ARRP probands, mutations associated with disease have been found in several candidate genes expressed in rod photoreceptors at very low frequency:  RHO , encoding rhodopsin, 1/126 patients screened;  PDE6B (click to continue reading) Based on average estimates of the prevalence of non-syndromic retinitis pigmentosa (RP) at 1/4,000, there are approximately 1.5 million people in the world with this inherited, progressive, degenerative disease of the retinal photoreceptor cells which often leads to blindness (1,2,3). About 50% of these cases are inherited in an autosomal recessive manner (AR). With the approach of screening the exons of candidate genes in large numbers of unrelated ARRP probands, mutations associated with disease have been found in several candidate genes expressed in rod photoreceptors at very low frequency: RHO , encoding rhodopsin, 1/126 patients screened (4); PDE6B , encoding the beta-subunit of rod cGMP-phosphodiesterase, 4/88 patients (5); PDE6A , encoding the alpha-subunit of rod cGMP-phosphodiesterase, 2/173 patients (6); and CNCG , encoding the alpha-subunit of the rod cGMP-gated channel, 3/173 patients (7). With the approach of linkage analysis in large families, two ARRP loci have so far been discovered: 1q31-q32.1 (8,9) and 6p, distal to RDS-peripherin (10). Also, linkage analysis of an informative consanguineous family led to the discovery of a second homozygous RHO mutation (11). ARRP tends to appear most often in small nuclear families that by themselves are not informative enough to yield significant linkage data, and screening all the exons of candidate genes like PDE6A and PDE6B (each with 22 exons) in large numbers of unrelated probands is costly and time consuming. Therefore, we have taken the approach of studying candidate genes in small nuclear ARRP families with a double screening protocol. Linkage analyses of markers close to the loci of the candidate genes are performed first, and any families where a gene locus clearly does not segregate with disease are ruled out from further study of that gene. DNAs of the probands from the remaining families (where the gene locus cannot be ruled out from segregating with disease) are then screened for mutations in the exons of the candidate gene by SSCPE (single strand conformation polymorphism electrophoresis) and DGGE (denaturing gradient gel electrophoresis). Any exonic variants found are sequenced directly and analyzed within the corresponding family to see if they appear to segregate with disease. With this approach we studied 24 families with inherited retinal degenerations (14 with typical RP) for mutations in the genes PDE6B, MYL5 , PDE6C, CNCG , RHO, ROM1 and RDS-peripherin . We have reported two typical ARRP families where the affecteds uniquely inherited compound heterozygous mutations in the PDE6B gene (12). With a similar approach, homozygous mutations also have been found in PDE6B in the affecteds of two other ARRP families (13,14). Click here to read the entire article

Richard J. Davis, Chun-Wei Hsu, Yi-Ting Tsai, Katherine J. Wert, Javier Sancho-Pelluz, Chyuan-Sheng Lin and Stephen H. Tsang | Journal of Neuroscience|  14 Aug 2013 | 33 (33) | 13475-13483 | doi.org/10.1523/JNEUROSCI.0419-13.2013 The third-most common cause of autosomal recessive retinitis pigmentosa (RP) is due to defective cGMP phosphodiesterase-6 (PDE6). Previous work using viral gene therapy on PDE6-mutant mouse models demonstrated photoreceptors can be rescued if administered before degeneration. However, whether visual function can be rescued after degeneration onset has not been addressed. This is a clinically important question, as newly diagnosed patients exhibit considerable loss of rods and cones in their peripheral retinas. We have generated and characterized a tamoxifen inducible Cre-loxP rescue allele, PDE6B. click here to read entire article.

S. Goyal ,  M. Jäger ,  P.N. Robinson ,  V. Vanita | Clinical Genetics | 21 July 2015 | Vol. 89, Issue 4 | pgs. 454-460 | https://doi.org/10.1111/cge.12644 Abstract Nonsyndromic retinitis pigmentosa (RP) is genetically highly heterogeneous, with >100 disease genes identified. However, mutations in these genes explain only 60% of all RP cases. Blood samples were collected from 12 members of an autosomal recessive RP family. Whole genome homozygosity mapping and haplotype analysis placed the RP locus in this family at chromosome 14q31.3. Whole-exome sequencing (WES) in proband revealed a mutation in TTC8 , which was flagged as most likely candidate gene by bioinformatic analysis. TTC8 is mutated in Bardet–Biedl syndrome 8 (BBS8), and once reported previously in a family with nonsyndromic RP. Sequencing of amplified products of exon 13 of TTC8 validated c.1347G>C (p.Gln449His), a novel change that affects the final nucleotide of exon 13 and might deleteriously affect splicing. This mutation segregated completely with the disease in the family and was not observed in 100 ethnically matched controls from same population. This represents second report of a TTC8 mutation in nonsyndromic RP, thus confirming the identity of TTC8 as causative gene for RP51. Click here to buy article

Michael Danciger, Vickie Heilbron, Yong-Qing Gao, Dan-Yun Zhao, Samuel G. Jacobson, and Debora B. Farber | Mol. Vis. | Volume 2 (10) | 1996 |  molvis.org/molvis/v2/a10/ Based on average estimates of the prevalence of non-syndromic  retinitis pigmentosa  (RP) at 1/4,000, there are approximately 1.5 million people in the world with this inherited, progressive, degenerative disease of the retinal photoreceptor cells which often leads to blindness. About 50% of these cases are inherited in an autosomal recessive manner (AR). With the approach of screening the exons of candidate genes in large numbers of unrelated ARRP probands, mutations associated with disease have been found in several candidate genes expressed in rod photoreceptors at very low frequency:  RHO , encoding rhodopsin, 1/126 patients screened;  PDE6B (click to continue reading) Based on average estimates of the prevalence of non-syndromic retinitis pigmentosa (RP) at 1/4,000, there are approximately 1.5 million people in the world with this inherited, progressive, degenerative disease of the retinal photoreceptor cells which often leads to blindness (1,2,3). About 50% of these cases are inherited in an autosomal recessive manner (AR). With the approach of screening the exons of candidate genes in large numbers of unrelated ARRP probands, mutations associated with disease have been found in several candidate genes expressed in rod photoreceptors at very low frequency: RHO , encoding rhodopsin, 1/126 patients screened (4); PDE6B , encoding the beta-subunit of rod cGMP-phosphodiesterase, 4/88 patients (5); PDE6A , encoding the alpha-subunit of rod cGMP-phosphodiesterase, 2/173 patients (6); and CNCG , encoding the alpha-subunit of the rod cGMP-gated channel, 3/173 patients (7). With the approach of linkage analysis in large families, two ARRP loci have so far been discovered: 1q31-q32.1 (8,9) and 6p, distal to RDS-peripherin (10). Also, linkage analysis of an informative consanguineous family led to the discovery of a second homozygous RHO mutation (11). ARRP tends to appear most often in small nuclear families that by themselves are not informative enough to yield significant linkage data, and screening all the exons of candidate genes like PDE6A and PDE6B (each with 22 exons) in large numbers of unrelated probands is costly and time consuming. Therefore, we have taken the approach of studying candidate genes in small nuclear ARRP families with a double screening protocol. Linkage analyses of markers close to the loci of the candidate genes are performed first, and any families where a gene locus clearly does not segregate with disease are ruled out from further study of that gene. DNAs of the probands from the remaining families (where the gene locus cannot be ruled out from segregating with disease) are then screened for mutations in the exons of the candidate gene by SSCPE (single strand conformation polymorphism electrophoresis) and DGGE (denaturing gradient gel electrophoresis). Any exonic variants found are sequenced directly and analyzed within the corresponding family to see if they appear to segregate with disease. With this approach we studied 24 families with inherited retinal degenerations (14 with typical RP) for mutations in the genes PDE6B, MYL5 , PDE6C, CNCG , RHO, ROM1 and RDS-peripherin . We have reported two typical ARRP families where the affecteds uniquely inherited compound heterozygous mutations in the PDE6B gene (12). With a similar approach, homozygous mutations also have been found in PDE6B in the affecteds of two other ARRP families (13,14). Click here to read more References Kumar-Singh, R, Farrar GJ, Mansergh, F. Kenna, P. Bhattacharya, S, Gal, A, and Humphries, P, Exclusion of the involvement of all known retinitis pigmentosa loci in the disease present in a family of Irish origin provides evidence for a sixth autosomal dominant locus (RP8). Hum. Mol. Genet. 7 (1993) 875-878 . Bundey, S and Crews, SSJ, A study of retinitis pigmentosa in the City of Birmingham. I Prevalence. J. Med. Genet. 21 (1984) 417-420. Boughman, JA, Conneally, PM and Nance WE, Population genetic studies of retinitis pigmentosa. Am. J. Hum. Genet. 32 (1980) 223-235. Rosenfeld, PJ, Cowley, GS, McGee, TL, Sandberg, MA, Berson, EL and Dryja, TP, A null mutation in the rhodopsin gene causes rod photoreceptor dysfunction and autosomal recessive retinitis pigmentosa. Nature Genet. 1 (1992) 209-213. McLaughlin, ME, Sandberg, MA, Berson, EL and Dryja TP, Recessive mutations in the gene encoding the beta-subunit of rod phosphodiesterase in patients with retinitis pigmentosa. Nature Genet. 4 (1993) 130-134. Huang, SH, Pittler, SJ, Huang, X, Oliveira, L, Berson, EL and Dryja, TP, Autosomal recessive retinitis pigmentosa caused by mutations in the a subunit of rod cGMP phosphodiesterase. Nature Genet. 11 (1995) 468-471. Dryja, TP, Finn, JT, Peng, Y-W, McGee, TL, Berson, EL and Yau, K-W, Mutations in the gene encoding the a subunit of the rod cGMP-gated channel in autosomal recessive retinitis pigmentosa. Proc. Nat. Acad. Sci. USA 92 (1995) 10177-10181. van Soest, S, Ingeborgh van den Born, L, Gal, A, Farrar, GJ, Bleeker- Wagemakers, LM, Westerveld, A, Humphries, P, Sandkuijl, LA and Bergen, AB, Assignment of a gene for autosomal recessive retinitis pigmentosa (RP12) to chromosome 1q31-q32.1 in an inbred and genetically heterogeneous disease population. Genomics 22 (1994) 499-504. Leutelt, J, Oehlman, R, Younus, F, Ingeborgh van den Born, L, Weber, JL, Denton, MJ, Qasim Mehdi, S and Gal, A, Autosomal recessive retinitis pigmentosa locus maps on chromosome 1q in a large consanguineous family from Pakistan. Clin. Genet. 47 (1995) 122-124. Knowles, JA, Shugart, Y, Banerjee, P, Gilliam, TC, Lewis, CA, Jacobson, SG and Ott, J, Identification of a locus, distinct from RDS-peripherin, for autosomal recessive retinitis pigmentosa on chromosome 6p. Hum. Mol. Genet. 3 (1994) 1402-1403. Kumaramanickavel, G, Maw, M, Denton, MJ, John, S, Srikumari, CR, Orth, U, Oehlman, R and Gal, A, Missense rhodopsin mutation in a family with recessive RP. Nature Genet. 8 (1994) 10-11. Danciger, M, Blaney, J, Gao, Y-Q, Zhao, DY, Heckenlively, JR, Jacobson, SG and Farber, DB, Mutations in the PDE6B gene in autosomal recessive RP. Genomics 30 (1995) 1-7. Bayes, M, Giordano, M, Balcells, S, Grinberg, S, Vilageliu, L, Martinez, I, Ayuso, C, Benitez, J, Ramos-Arroyo, MA, Chivelet, P, Solans, T, Valverde, D, Amselem, S, Goosens, M, Baiget, M, Gonzalez-Duarte, R and Besmond, C, Homozygous tandem duplication within the gene encoding the beta-subunit of rod phosphodiesterase as a cause for autosomal recessive retinitis pigmentosa. Human Mutation 5 (1995) 228-234. Valverde, D, Solans, T, Grinberg, S, Balcells, S,Vilageliu, L, Bayes, M, Chivelet, P, Besmond, C, Goosens, Gonzalez-Duarte, R and Baiget, M, A novel mutation in exon 17 of the beta-subunit of rod phosphodiesterase in two RP sisters of a consanguineous family. Human Genet. 97 (1996) 35-38.

Dunja Lukovic , Ana Artero Castro , Ana Belen Garcia Delgado , María de los Angeles Martín Bernal , Noelia Luna Pelaez , Andrea Díez Lloret , Rocío Perez Espejo , Kunka Kamenarova , Laura Fernández Sánchez , Nicolás Cuenca , Marta Cortón , Avila Fernandez , Anni Sorkio , Heli Skottman , Carmen Ayuso , Slaven Erceg , Shomi S. Bhattacharya  | Scientific Reports | Human iPSC derived disease model of MERTK-associated retinitis pigmentosa | 11 August 2015 | https://doi.org/10.1038/srep12910 Abstract Retinitis pigmentosa (RP) represents a genetically heterogeneous group of retinal dystrophies affecting mainly the rod photoreceptors and in some instances also the retinal pigment epithelium (RPE) cells of the retina. Clinical symptoms and disease progression leading to moderate to severe loss of vision are well established and despite significant progress in the identification of causative genes, the disease pathology remains unclear. Lack of this understanding has so far hindered development of effective therapies. Here we report successful generation of human induced pluripotent stem cells (iPSC) from skin fibroblasts of a patient harboring a novel Ser331Cysfs*5 mutation in the MERTK gene. The patient was diagnosed with an early onset and severe form of autosomal recessive RP (arRP). Upon differentiation of these iPSC towards RPE, patient-specific RPE cells exhibited defective phagocytosis, a characteristic phenotype of MERTK deficiency observed in human patients and animal models. Thus we have created a faithful cellular model of arRP incorporating the human genetic background which will allow us to investigate in detail the disease mechanism, explore screening of a variety of therapeutic compounds/reagents and design either combined cell and gene- based therapies or independent approaches. Introduction Retinitis pigmentosa (RP; OMIM 268000) with a prevalence of 1 in 3,500 individuals is the most common form of hereditary retinal disorder affecting the working age group. RP is characterized by progressive dysfunction and death of mainly the rod photoreceptor cells (PR) of the retina however in some cases retinal pigment epithelium (RPE) cells are also involved, often resulting in permanent blindness. So far 54 genes have been implicated in this disease coding for proteins involved in a myriad of functions such as phototransduction signaling cascade, retinoid cycle, cell-cell adhesion or the cytoskeleton 1 . The disease is inherited in all there Mendelian forms, the autosomal recessive (arRP) being the most common with over 50% of cases. Largely due to the high genetic heterogeneity and unavailability of disease tissue, pathology of the disease remains elusive. Patient-derived induced pluripotent stem cells (iPSCs) provide an unprecedented opportunity to recapitulate disease pathogenicity without the need for genetic manipulation and creation of gene targeted animal models. Human iPSCs, similar to embryonic stem cells (ESC), can be expanded indefinitely in vitro and differentiated into any type of mature cell in the human body, without the ethical and immunogenicity issues associated with ESC 2 . These cells are also valuable for developing therapeutic strategies, drug toxicity screens and development of disease models, in addition to providing a source for cell transplantation therapy. RPE cells and photoreceptors (PR) have been successfully generated from iPSCs (iPSC-RPE and iPSC-PR respectively) by various groups in stepwise differentiation protocols mimicking retinal development by introducing Wnt signaling inhibitors (DKK1), Nodal antagonist Lefty A, Notch pathway inhibitor (DAPT-gamma secretase inhibitor), or IGF-1 3 , 4 , 5 . In contrast, only RPE cells have been generated spontaneously in overgrown iPSC/ESC cultures without the addition of exogenous factors, since derivatives of neuroectoderm appear by default in non-induced cultures 6 , 7 . Generated RPE cells in these studies display a fully mature phenotype and physiological activity in vitro such as phagocytosis, secretion of vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF) and epithelial barrier formation. Cellular models of hereditary retinal dystrophies have been successfully created in vitro in Best disease and RP where patients’ fibroblasts were reprogrammed to iPSC and then converted to RPE 8 , 9 or photoreceptor cells 10 , expressing the disease phenotype. iPSC- derived RPE (iPSC-RPE) cells have also been shown to have a protective effect when injected sub-retinaly into the Royal College of Surgeons (RCS) rats 11 and RPE65-defective mice 12 . Moreover, iPSCs have met clinical-grade requirements 13 as a source of RPE grafts and have recently been injected in patients affected by the exudative form (wet-type) of age-related macular degeneration (AMD) 14 . It has been argued that in this form of AMD the dysfunction and loss of RPE cells is the main cause of visual impairment in the elderly. Mer tyrosine kinase receptor (MERTK) belongs to the Tyro3/Axl/Mer (TAM) receptor tyrosine kinase family of proteins distinguished by a conserved intracellular kinase domain and extracellular adhesion molecule-like domain. TAM receptors regulate a variety of processes such as cell proliferation/survival, adhesion, migration, inflammatory response, in a cell- microenvironment- and ligand- specific manner 15 . In previous studies MERTK was found to be disrupted in RCS rats 16 , 17 , a classic model for retinal degeneration inherited as an autosomal recessive trait and found to cause early-onset retinitis pigmentosa in patients 18 . RPE cells fail to phagocytize the shed outer segment (OS) material of PR, a circadian activity performed by RPE cells which serves to renew the damaged lipid and protein components of light exposed PR, while new membranous discs are formed (disc biogenesis) and inserted in the basal part of the OS. As a result, RCS rats exhibit OS associated debris accumulation in the subretinal space, abnormal OS length, eventually leading to the onset of PR degeneration by the P20 stage. Usually complete degeneration occurs by P60. Similar phenotype is observed in merkd mice 19 indicating that the RPE phagocytic defect is the underlying molecular mechanism of disease in humans carrying MERTK mutations. Indeed, the reduced retinal thickness and debris detected in the sub-retinal space in patients harboring the MERTK –splice-site -mutation resembles the observed phenotype in the RCS rat 20 . The distinctive clinical presentation of RP is the only disease manifestation of patients harboring MERTK mutations without any systemic disease or defects of phagocytosis by macrophages, indicating a specialized function of this protein in the RPE cells. In contrast to the detailed clinical understanding of the disease, the mechanism by which MERTK acts during the phagocytosis remains partially unveiled. Outer segments are known to bind to the integrin receptor αvβ5 21 followed by focal adhesion kinase (FAK) activation in the apical membrane of RPE 22 while the MERTK activation occurs via Gas6/Protein S, TUB, TULP1 ligand binding 23 , 24 . The latter is thought to activate autophosphorylation at tyrosine Y-749, Y-753 and Y-754 in the tyrosine kinase domain, which in turn activates the molecular cascade targeting actin or non-muscle myosin II to coordinate the cytoskeletal rearrangements necessary for phagocytic ingestion 25 . Click here to read entire article References Hartong, D. 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Camiel J F Boon   1 ,  B Jeroen Klevering ,  Bart P Leroy ,  Carel B Hoyng ,  Jan E E Keunen ,  Anneke I den Hollander | Prog Retin Eye Res | 2009 May |28(3) | Pages 187-205 | doi: 10.1016/j.preteyeres.2009.04.002 Abstract Bestrophin-1 is an integral membrane protein, encoded by the BEST1 gene, which is located in the basolateral membrane of the retinal pigment epithelium. The bestrophin-1 protein forms a Ca(2+) activated Cl(-) channel and is involved in the regulation of voltage-dependent Ca(2+) channels. In addition, bestrophin-1 appears to play a role in ocular development. Over 120 different human BEST1 mutations have been described to date, associated with a broad range of ocular phenotypes. The purpose of this review is to describe this spectrum of phenotypes, which includes Best vitelliform macular dystrophy and adult-onset foveomacular vitelliform dystrophy, autosomal dominant vitreoretinochoroidopathy, the MRCS (microcornea, rod-cone dystrophy, cataract, posterior staphyloma) syndrome, and autosomal recessive bestrophinopathy. The genotype-phenotype correlations that are observed in association with BEST1 mutations are discussed. In addition, in vitro studies and animal models that clarify the pathophysiological mechanisms are reviewed. Introduction Best vitelliform macular dystrophy (BVMD) is among the most frequently encountered autosomal dominant (AD) retinal dystrophies and predominantly affects the macula. BVMD was the first disease shown to be caused by mutations in the BEST1 gene, which encodes the bestrophin-1 protein that localizes to the retinal pigment epithelium (RPE) (Petrukhin et al., 1998). Subsequent studies showed that BEST1 gene mutations may also be found in patients with adult-onset foveomacular vitelliform dystrophy (AFVD) (Allikmets et al., 1999, Kramer et al., 2000, Seddon et al., 2001). BVMD and AFVD are related phenotypes with abnormalities that are generally restricted to the macula. However, more widespread ocular abnormalities may arise in association with specific BEST1 gene mutations that cause AD vitreoretinochoroidopathy (ADVIRC) and AD MRCS (microcornea, rod-cone dystrophy, early-onset cataract, and posterior staphyloma) syndrome (Reddy et al., 2003, Yardley et al., 2004, Michaelides et al., 2006, Burgess et al., 2008a). The same applies to autosomal recessive bestrophinopathy (ARB), the human null phenotype of bestrophin-1, which is associated with high hyperopia and shallow anterior chambers (Burgess et al., 2008b). Therefore, ADVIRC, MRCS syndrome, as well as ARB belong to a spectrum of diseases with abnormal ocular development that extends beyond the retina. In this paper, we aim to review the characteristics of the BEST1 gene and its multifunctional protein product bestrophin-1, with an emphasis on the broad spectrum of ocular phenotypes associated with mutations in this gene. The effects of different BEST1 mutations are discussed, as well as their genotype–phenotype correlations. Available in vitro and animal models are addressed, as well as histopathologic observations in BEST1 -related diseases, that expand our insight in the pathogenesis. Finally, perspectives on future therapeutic strategies are discussed. The BEST1 gene The human BEST1 gene was identified in 1998 by Petrukhin and colleagues (Petrukhin et al., 1998). BEST1 is located on chromosome 11q12, spans 15 kilobases of genomic DNA and contains 11 exons of which 10 are protein-coding (Marquardt et al., 1998, Petrukhin et al., 1998). 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DeLuca, AP(DeLuca, Adam P.); Weed, MC(Weed, Matthew C.); Haas, CM(Haas, Christine M.); Halder, JA(Halder, Jennifer A.); Stone, EM(Stone, Edwin M.) | JAMA Ophthalmology | August 2015 | Vol. 133(8) | pgs. 967-968 | doi:10.1001/jamaophthalmol.2015.1463 The clinical features of a known mendelian disease can occasionally be mimicked by the random co-occurrence of 2 different conditions in the same individual. We report a case in which whole-exome sequencing in a patient previously suspected to have Usher syndrome revealed disease-causing mutations in BBS1 and SLC26A4 . This case illustrates how detailed and accurate clinical data are needed to interpret exome-scale genetic testing results. A man in his late 30s was referred for evaluation of suspected Usher syndrome. As a toddler, he was diagnosed as having sensorineural hearing loss due to a Mondini malformation after computed tomography showed characteristic cochlear abnormalities (audiograms shown in Figure 1 ). A few years later, his parents noted that he began tripping over objects and having trouble seeing at night. Several years later, he was diagnosed as having retinitis pigmentosa and suspected to have Usher syndrome. At that time, his best-corrected visual acuity was 20/60 OD and 20/80 OS. By his early 20s, it had decreased to counting fingers OD and 20/800 OS. Aside from a paternal great-great-aunt with deafness, mutism, and normal vision, there were no other affected family members. To buy article, click here