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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

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). Eight years later, the mouse ortholog was characterized (Kramer et al., 2004). An alternative name for BEST1 is VMD2 , but the Human Genome Organisation and the Mouse Genome Database nomenclature committees recently recommended Click here to read entire article References R.E. Andrade et al. Photodynamic therapy with verteporfin for subfoveal choroidal neovascularization in Best disease Am. J. Ophthalmol. (2003) B. Bakall et al. Enhanced accumulation of A2E in individuals homozygous or heterozygous for mutations in BEST1 (VMD2) Exp. Eye Res. (2007) R. Barro Soria et al. Bestrophin 1 and 2 are components of the Ca(2+) activated Cl(−) conductance in mouse airways Biochim. Biophys. Acta (2008) N. Benhamou et al. Adult-onset foveomacular vitelliform dystrophy: a study by optical coherence tomography Am. J. Ophthalmol. (2003) W.E. Benson et al. Best's vitelliform macular dystrophy Am. J. Ophthalmol. (1975) C.J.F. Boon et al. The spectrum of retinal dystrophies caused by mutations in the peripherin/RDS gene Prog. Retin. Eye Res. (2008) C.J.F. Boon et al. Fundus autofluorescence imaging of retinal dystrophies Vis. Res. (2008) C.J.F. Boon et al. Basal laminar drusen caused by compound heterozygous variants in the CFH gene Am. J. Hum. Genet. (2008) D.B. Burgess et al. Macular disease resembling adult foveomacular vitelliform dystrophy in older adults Ophthalmology (1987) R. Burgess et al. Biallelic mutation of BEST1 causes a distinct retinopathy in humans Am. J. Hum. Genet. (2008) G.M. Caldwell et al. Bestrophin gene mutations in patients with Best vitelliform macular dystrophy Genomics (1999) L. Cartegni et al. Determinants of exon 7 splicing in the spinal muscular atrophy genes, SMN1 and SMN2 Am. J. Hum. Genet. (2006) S.Y. Cohen et al. Monozygotic twin sisters with adult vitelliform macular dystrophy Am. J. Ophthalmol. (1993) J. Dillon et al. The photochemical oxidation of A2E results in the formation of a 5,8,5',8'-bis-furanoid oxide Exp. Eye Res. (2004) N. Esumi et al. Analysis of the VMD2 promoter and implication of E-box binding factors in its regulation J. Biol. Chem. (2004) N. Esumi et al. VMD2 promoter requires two proximal E-box sites for its activity in vivo and is regulated by the MITF-TFE family J. Biol. Chem. (2007) G.A. Fishman et al. Visual acuity in patients with Best vitelliform macular dystrophy Ophthalmology (1993) T. Furukawa et al. Crx , a novel otx-like homeobox gene, shows photoreceptor-specific expression and regulates photoreceptor differentiation Cell (1997) R.P. Gallemore et al. Retinal pigment epithelial transport mechanisms and their contributions to the electroretinogram Prog. Retin. Eye Res. (1997) M.F. Goldberg et al. Histopathologic study of autosomal dominant vitreoretinochoroidopathy. Peripheral annular pigmentary dystrophy of the retina Ophthalmology (1989) J.S. Adorante et al. Potassium-dependent volume regulation in retinal pigment epithelium is mediated by Na, K, Cl cotransport J. Gen. Physiol. (1990) J.J. Alexander et al. Adeno-associated viral vectors and the retina Adv. Exp. Med. Biol. (2008) R. Allikmets et al. Evaluation of the Best disease gene in patients with age-related macular degeneration and other maculopathies Hum. Genet. (1999) R.E. Andrade et al. Optical coherence tomography in choroidal neovascular membrane associated with Best's vitelliform dystrophy Acta Ophthalmol. Scand. (2002) S.L. Ang et al. A targeted mouse Otx2 mutation leads to severe defects in gastrulation and formation of axial mesoderm and to deletion of rostral brain Development (1996) M.A. Apushkin et al. Novel de novo mutation in a patient with Best macular dystrophy Arch. Ophthalmol. (2006) J.J. Arnold et al. Adult vitelliform macular degeneration: a clinicopathological study Eye (2003) L.O. Atchaneeyasakul et al. Mutation analysis of the VMD2 gene in Thai families with Best macular dystrophy Ophthalmic Genet. (2008) W. Baca et al. Dark adaptation in patients with Best vitelliform macular dystrophy Br. J. Ophthalmol. (1994) J.W. Bainbridge et al. Gene therapy progress and prospects: the eye Gene Ther. (2006) B. Bakall et al. The mutation spectrum of the bestrophin protein-functional implications Hum. Genet. (1999) B. Bakall et al. Expression and localization of bestrophin during normal mouse development Invest. Ophthalmol. Vis. Sci. (2003) B. Bakall et al. Bestrophin-2 is involved in the generation of intraocular pressure Invest. Ophthalmol. Vis. Sci. (2008) D.B. Barr et al. Autofluorescence in a patient with adult vitelliform degeneration Eur. J. Ophthalmol. (1995) R. Barro Soria et al. Bestrophin 1 enables Ca2+ activated Cl− conductance in epithelia J. Biol. Chem. (2006) M. Battaglia Parodi et al. Vascularized pigment epithelial detachment in adult-onset foveomacular vitelliform dystrophy Eur. J. Ophthalmol. (2000) M. Battaglia Parodi et al. Photodynamic therapy for choroidal neovascularization associated with pattern dystrophy Retina (2003) C. Baum et al. Mutagenesis and oncogenesis by chromosomal insertion of gene transfer vectors Hum. Gene Ther. (2006) F. Best Über eine hereditäre Maculaaffektion. Beitrag zur Vererbungslehre Z. Augenheilk (1905) S. Bialek et al. K+ and Cl− transport mechanisms in bovine pigment epithelium that could modulate subretinal space volume and composition J. Physiol. (1994) C.F. Blodi et al. Best's vitelliform dystrophy Ophthalmic Paediatr. Genet. (1990) L.H. Bloom et al. Adult vitelliform macular degeneration Br. J. Ophthalmol. (1981) C.J.F. Boon et al. Clinical and genetic heterogeneity in multifocal vitelliform dystrophy Arch. Ophthalmol. (2007) Boon, C.J.F., Theelen, T., Hoefsloot, E.H., van Schooneveld, M.J., Keunen, J.E.E., Cremers, F.P.M., Klevering, B.J.,... R. Brecher et al. Adult vitelliform macular dystrophy Eye (1990) N.M. Bressler et al. Natural course of poorly defined choroidal neovascularization associated with macular degeneration Arch. Ophthalmol. (1988) P.K. Buch et al. AAV-mediated gene therapy for retinal disorders: from mouse to man Gene Ther. (2008) K.M. Bumsted et al. Dorsal retinal pigment epithelium differentiates as neural retina in the microphthalmia (mi/mi) mouse Invest. Ophthalmol. Vis. Sci. (2000) R. Burgess et al. ADVIRC is caused by distinct mutations in BEST1 that alter pre-mRNA splicing J. Med. Genet. (2008) M. Caputi et al. A novel bipartite splicing enhancer modulates the differential processing of the human fibronectin EDA exon Nucleic Acids Res. (1994)

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.

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.

Muhammad Imran Khan, Ferry Kersten, Maleeha Azam, Rob Collin, Alamdar Hussain, Syed Tahir-A Shah, Jan Keunen, Hannie Kremer, Frans Cremers, Raheel Qamar, Anneke den Hollander |  Ophthalmology​ | 2011 Jul 1 | Vol.18, Issue 7 |1444-8 | doi: 10.1016/j.ophtha.2010.10.047    Objective: To describe the mutations in the CLRN1 gene in patients from 2 consanguineous Pakistani families diagnosed with autosomal recessive retinitis pigmentosa (arRP). Participants: Affected and unaffected individuals of 2 consanguineous Pakistani families and 90 unaffected controls from the same population. Informed consent was obtained from participants and the protocol was approved by a local institutional review board.

Bernardo V Alvarez, Eranga N Vithana, Zhenglin Yang, Adrian H Koh, Kit Yeung, Victor Yong, Haley J Shandro, Yali Chen, Prasanna Kolatkar, Paaventhan Palasingam, Kang Zhang, Tin Aung, Joseph R Casey | Investigative Ophthalmology & Visual Science  | Aug 2007 | Vol.48 | 3459-3468 | doi.org/10.1167/iovs.06-1515 Abstract Purpose The autosomal dominant retinitis pigmentosa (adRP) gene on chromosome 17, region q22 (RP17), was recently identified as a glycosylphosphatidylinositol membrane-anchored zinc metalloenzyme (protein CAIV), highly expressed in the choriocapillaris of the eye and undetectable in the retina. Only two missense mutations have thus far been identified in the gene CA4 . Functional analysis of these mutations demonstrated that retinal disease may result from perturbation of pH homeostasis in the outer retina, after disruption of CAIV and sodium bicarbonate cotransporter 1 (NBC1)–mediated bicarbonate transport. CA4 was screened in a panel of patients with RP, to expand the mutation spectrum of this novel adRP gene and understand its pathogenic mechanism. Methods A total of 96 patients with simplex RP and adRP of Chinese ethnicity were screened for mutations in the eight coding exons of the CA4 gene by bidirectional sequencing. Functional consequences of CA4 mutations on the NBC1-mediated bicarbonate transport were studied by measuring bicarbonate fluxes in HEK293 cells cotransfected with NBC1 and CA4 mutant cDNAs. Results Thirteen sequence alterations were identified, including a novel mutation within exon 3 of CA4 (R69H) in a patient with simplex RP. R69H was not found in 432 normal chromosomes. R69H CAIV impaired NBC1-mediated pH recovery after acid load. Conclusions A novel mutation has been identified in CA4 that provides further evidence that impaired pH regulation may underlie photoreceptor degeneration in RP17. This study indicates that, as with European patients with RP, mutations in CA4 also account for ≤1% of Chinese patients with RP. Click here to read original article

Aisha Pandor, Rajkumar Ramesar, Sharon Prince | J Cell Biochem ​ | 2010 Oct 15 | Vol. 111, Issue 3 | 735-41 |  doi: 10.1002/jcb.22759 Abstract Retinitis pigmentosa is a highly heterogeneous form of inherited blindness which affects more than 1.3 million individuals worldwide. The RP17 form of the disease is caused by an arginine to tryptophan (R14W) mutation in the signal sequence of carbonic anhydrase IV (CAIV). While CAIV is expressed in the choriocapillaries of the eye and renal epithelium, the R14W mutation results in an exclusively ocular phenotype in affected individuals. In order to investigate the mechanism of disease in RP17 and the lack of kidney phenotype, we compared the subcellular localization and post-translational processing of wild-type (WT)- and mutant-CAIV in three cell types. We show using immunocytochemistry that unlike WT CAIV which is transported to the plasma membrane of transfected COS-7 and HT-1080 cells, the R14W mutant CAIV is retained in the endoplasmic reticulum. Western blot analyses further reveal that whereas the WT CAIV is processed to its mature form in both these cell lines, significant levels of the R14W mutant protein remain in its immature form. Importantly, flow cytometry experiments demonstrate that compared to WT CAIV protein, expression of specifically the R14W CAIV results in an S and G2/M cell-cycle block, followed by apoptosis. Interestingly, when the above experiments were repeated in the human embryonic kidney cell line, HEK-293, strikingly different results were obtained. These cells were unaffected by the expression of the R14W mutant CAIV and were able to process the mutant and WT protein equally effectively. This study has important implications for our understanding of the RP17 phenotype. To read full text, click here

Lina Zelinger , Eyal Banin, Alexey Obolensky, Liliana Mizrahi-Meissonnier, Avigail Beryozkin, Dikla Bandah-Rozenfeld, Shahar Frenkel, Tamar Ben-Yosef, Saul Merin,  Sharon B. Schwartz ,  Artur V. Cideciyan , Samuel G. Jacobson, Dror Sharon |   American Society of Human Genetics (AJHG) |  2011 Feb 11 | 88(2) | 207–215 |  doi: 10.1016/j.ajhg.2011.01.002 Abstract Retinitis pigmentosa (RP) is a heterogeneous group of inherited retinal degenerations caused by mutations in at least 50 genes. Using homozygosity mapping in Ashkenazi Jewish (AJ) patients with autosomal-recessive RP (arRP), we identified a shared 1.7 Mb homozygous region on chromosome 1p36.11. Sequence analysis revealed a founder homozygous missense mutation, c.124A>G (p.Lys42Glu), in the dehydrodolichyl diphosphate synthase gene ( DHDDS ) in 20 AJ patients with RP of 15 unrelated families. The mutation was not identified in an additional set of 109 AJ patients with RP, in 20 AJ patients with other inherited retinal diseases, or in 70 patients with retinal degeneration of other ethnic origins. The mutation was found heterozygously in 1 out of 322 ethnically matched normal control individuals. RT-PCR analysis in 21 human tissues revealed ubiquitous expression of DHDDS . Immunohistochemical analysis of the human retina with anti-DHDDS antibodies revealed intense labeling of the cone and rod photoreceptor inner segments. Clinical manifestations of patients who are homozygous for the c.124A>G mutation were within the spectrum associated with arRP. Most patients had symptoms of night and peripheral vision loss, nondetectable electroretinographic responses, constriction of visual fields, and funduscopic hallmarks of retinal degeneration. DHDDS is a key enzyme in the pathway of dolichol, which plays an important role in N -glycosylation of many glycoproteins, including rhodopsin. Our results support a pivotal role of DHDDS in retinal function and may allow for new therapeutic interventions for RP. Main Text Retinitis pigmentosa (RP; MIM 268000 ) is the most common inherited retinal degeneration, with an estimated worldwide prevalence of 1:4000. 1–3 The disease is highly heterogeneous and has several patterns of inheritance. At present, 35 genetic loci have been implicated in nonsyndromic autosomal-recessive RP (arRP), most of which account for a few percent of RP cases each. Although many of the early identified arRP genes were excellent candidates for the disease when mutated, mainly because of the function of the encoded protein (e.g., PDE6A 4 [MIM 180071 ] and PDE6B 5 [MIM 180072 ]), a large proportion of the recently identified genes were not a priori considered as candidates and were identified through whole-genome linkage or homozygosity mapping followed by mutation screening of a large number of genes in the linked intervals (e.g., EYS 6 [MIM 612424 ] and SPATA7 7 [MIM 609868 ]). Through those studies, a new class of genes encoding proteins with housekeeping-like function (e.g., IDH3B [MIM 604526 ] for arRP 8 and splicing factors for autosomal-dominant RP 9,10 ) have been identified and provided new insight into processes that result in retinal degeneration. The reason for the retina-specific phenotype caused by mutations in these genes is still unclear. The Ashkenazi Jewish (AJ) population was established by Jews who originated in the Middle East and migrated to Europe, initially settling in Germany (the “Ashkenaz” region) at or before the 4 th century. The AJ lived in closed communities in European countries and developed a unique culture and language (named Yiddish, which is based on a few different languages, including German, Hebrew, and Aramaic). After the Holocaust, the population size dropped from about 8.8 million individuals to only 2.8 million, and AJ immigrated out of Europe, mainly to the United States and the emerging state of Israel. AJ currently constitute the largest Jewish ethnic group in both countries. A large amount of effort was directed to study the genetic structure of the AJ population, in the context of other Jewish ethnic groups and Middle Eastern populations, at the Y chromosome, 11,12 mitochondrial, 13 and genomic 14,15 levels. Although consanguineous marriages are relatively uncommon among AJ (1.5% and rapidly declining), 16,17 most individuals who are affected by a rare AR disease in this ethnic group are homozygous for the disease-causing mutation, mainly because of a high rate of intracommunity marriages. 17 Therefore, genetic analysis of hereditary diseases in the AJ population, via homozygosity mapping, can be highly efficient. To read entire article, click here

Hiroyuki Morimura , Gerald A. Fishman, Sandeep A. Grover, Anne B. Fulton, Eliot L. Berson, Thaddeus P. Dryja |   PNAS | 17 May 1998 | Vol. 95 (6) | pgs. 3088–3093 | doi:  10.1073/pnas.95.6.3088 Abstract RPE65 is a protein of unknown function expressed specifically by the retinal pigment epithelium. We examined all 14 exons of this gene in 147 unrelated patients with autosomal recessive retinitis pigmentosa (RP), in 15 patients with isolate RP, and in 45 patients with Leber congenital amaurosis (LCA). Sequence anomalies that were likely to be pathogenic were found in two patients with recessive RP, in one patient with isolate RP recategorized as recessive, and in seven patients with LCA. Cosegregation analysis in each available family showed that all affected individuals were either homozygotes or compound heterozygotes and that all unaffected individuals were either heterozygote carriers or homozygous wild type. In one family, there was one instance of a new mutation not present in either parent of the affected individual. In another family, affected members with recessive RP in three branches (i.e., three distinct pairs of parents) were compound heterozygotes for the same two mutations or homozygous for one of them. Based on our results, mutations in the RPE65 gene appear to account for ≈2% of cases of recessive RP and ≈16% of cases of LCA. Over 40 loci for human hereditary retinal degenerations involving the photoreceptors and retinal pigment epithelium are known ( http://utsph.sph.uth.tmc.edu/www/utsph/RetNet/home.htm ). Most of these loci are unidentified genes that have been recognized by linkage studies alone. Our group is engaged in identifying genes responsible for this set of diseases primarily through a candidate gene-based approach ( 1 ). In this report, we describe an analysis of the RPE65 gene, which has been assigned to chromosome 1p31 ( 2 ). This gene was discovered through its protein product that forms a complex with an antibody raised against human retinal pigment epithelium ( 3 ). The cDNA sequence predicts a protein with 533 amino acid residues and a molecular mass of ≈61 kDa ( 4 ). The protein is associated with the endoplasmic reticulum of the retinal pigment epithelium in vertebrates ( 3 , 5 ). Although the biochemical function of the protein product is currently obscure, the tissue-specific expression of the RPE65 gene made it an attractive candidate as a cause for some retinal degenerations because the retinal pigment epithelium has an essential role in maintaining the viability of the neighboring photoreceptor cells. In particular, enzymes in the endoplasmic reticulum are responsible for the recycling of the chromophore used by photoreceptor opsins. To explore the possible role that defects in RPE65 might play in the etiology of retinal degenerations, we screened a large cohort of unrelated patients with retinitis pigmentosa or Leber congenital amaurosis for mutations. Because RPE65 is expressed specifically in the eye, the study focused on patients with nonsyndromic forms of these diseases. This study was carried out contemporaneously with studies from two other groups that recently have been published ( 6 , 7 ) . Click here to read entire article References Dryja T P. Proc Natl Acad Sci USA. 1997;94:12117–12121. doi: 10.1073/pnas.94.22.12117. Hamel C P, Jenkins N A, Gilbert D J, Copeland N G, Redmond T M. Genomics. 1994;20:509–512. doi: 10.1006/geno.1994.1212. Hamel C P, Tsilou E, Harris E, Pfeffer B A, Hooks J J, Detrick B, Redmond T M. J Neurosci Res. 1993;34:414–425. doi: 10.1002/jnr.490340406. Hamel C P, Tsilou E, Pfeffer B A, Hooks J J, Detrick B, Redmond T M. J Biol Chem. 1993;268:15751–15757. Nicoletti A, Wong D J, Kawase K, Gibson L H, Yang-Feng T L, Richards J E, Thompson D A. Hum Mol Genet. 1995;4:641–649. doi: 10.1093/hmg/4.4.641. Marlhens F, Bareil C, Griffoin J M, Zrenner E, Amalric P, Eliaou C, Liu S Y, Harris E, Redmond T M, Arnaud B, et al. Nat Genet. 1997;17:139–141. doi: 10.1038/ng1097-139. Gu S M, Thompson D A, Srikumari C R S, Lorenz B, Finckh U, Nicoletti A, Murthy K R, Rathmann M, Kumaramanickavel G, Denton M J, et al. Nat Genet. 1997;17:194–197. doi: 10.1038/ng1097-194.

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.

Isosomppi, J., Västinsalo, H., Geller, S. F., Heon, E., Flannery, J. G., & Sankila, E. M. | Molecular Vision | 2009 Sep 8 | Vol. 15 | pgs. 1806-18 | ncbi.nlm.nih.gov/pmc/articles/PMC2742642/ Abstract Purpose Mutations of clarin 1 ( CLRN1 ) cause Usher syndrome type 3 (USH3). To determine the effects of USH3 mutations on CLRN1 function, we examined the cellular distribution and stability of both normal and mutant CLRN1 in vitro. We also searched for novel disease-causing mutations in a cohort of 59 unrelated Canadian and Finnish USH patients. Methods Mutation screening was performed by DNA sequencing. For the functional studies, wild-type (WT) and mutant CLRN1 genes were expressed as hemagglutinin (HA) tagged fusion proteins by transient transfection of BHK-21 cells. Subcellular localization of CLRN1-HA was examined by confocal microscopy. The N-glycosylation status of CLRN1 was studied by using the N-glycosidase F (PNGase F) enzyme and western blotting. Cycloheximide treatment was used to assess the stability of CLRN1 protein. Results We found three previously reported pathogenic mutations, p.A123D, p.N48K, and p.Y176X, and a novel sequence variant, p.L54P, from the studied USH patients. The WT HA-tagged CLRN1 was correctly trafficked to the plasma membrane, whereas mutant CLRN1-HA proteins were mislocalized and retained in the endoplasmic reticulum. PNGase F treatment of CLRN1-HA resulted in an electrophoretic mobility shift consistent with sugar residue cleavage in WT and in all CLRN1 mutants except in p.N48K mutated CLRN1, in which the mutation abolishes the glycosylation site. Inhibition of protein expression with cycloheximide indicated that WT CLRN1-HA remained stable. In contrast, the CLRN1 mutants showed reduced stability. Conclusions WT CLRN1 is a glycoprotein localized to the plasma membrane in transfected BHK-21 cells. Mutant CLRN1 proteins are mislocalized. We suggest that part of the pathogenesis of USH3 may be associated with defective intracellular trafficking as well as decreased stability of mutant CLRN1 proteins. Introduction Usher syndrome (USH) describes a group of autosomal recessive diseases with bilateral sensorineural hearing loss and visual impairment phenotypically similar to retinitis pigmentosa (RP) [ 1 - 4 ]. Prevalence of USH in different populations is estimated to range from 3.5 to 6.2 per 100,000, thus making it the most frequent cause of combined deaf-blindness worldwide [ 5 ]. The condition has been classified into three clinical subtypes (USH1, USH2, and USH3), based on the severity and progression of the hearing impairment, presence or absence of vestibular dysfunction, and the age of onset of RP [ 1 ]. This classification remains in clinical use, although recent progress on the molecular genetics and clinical research of USH has revealed broad genetic and clinical heterogeneity [ 3 , 6 ]. Atypical forms of USH have been identified within all three clinical types, and there is considerable overlap of symptoms among the subtypes. A distinguishing feature of USH3 is the wide spectrum of nonlinear progressive hearing impairment, which ranges from a near normal to a severe audiometric phenotype [ 7 ]. USH3 patients may also have either normal or decreased vestibular responses [ 8 ]. The rate of visual loss in USH3 is similar to other USH subtypes [ 9 ], with the most recent analyses suggesting that retinal degeneration in USH3 progresses more rapidly than in USH2A [ 10 , 11 ]. The variable phenotype may cause USH3 to be under-diagnosed and it may be more prevalent than previously indicated [ 6 ]. To date, nine USH gene products have been identified: the molecular motor myosin VIIa (USH1B) [ 12 ]; the cell adhesion proteins cadherin 23 (USH1D) [ 13 ] and protocadherin 15 (USH1F) [ 14 , 15 ]; the scaffold proteins harmonin (USH1C) [ 16 ], SANS (USH1G) [ 17 ], and whirlin (USH2D) [ 18 ]; the G-protein-coupled 7-transmembrane receptor VLGR1b (USH2C) [ 19 ]; two isoforms of the extracellular matrix connected protein usherin (USH2A) [ 20 , 21 ]; and the four-pass transmembrane domain protein clarin 1 (USH3) [ 22 , 23 ]. There is growing evidence suggesting that these proteins form a network, which is critical for the development and maintenance of the sensorineural cells in the inner ear and the retina [ 3 , 4 , 24 - 28 ]. Click here to read original source article References Petit C. Usher syndrome: from genetics to pathogenesis. Annu Rev Genomics Hum Genet. 2001;2:271–97. doi: 10.1146/annurev.genom.2.1.271. Ahmed ZM, Riazuddin S, Wilcox ER. 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