Douglas Vollrath

Electric fields can be manipulated by a method in which multiple electrodes are arranged along a closed contour and clamped to predetermined electric potentials. This method may be applied to a broad range of problems in the separation of macromolecules by gel electrophoresis. DNA molecules as large as 2 megabases can be well separated with a contour-clamped homogeneous electric field alternating between two orientations 120 degrees apart. The pattern of separation is independent of position in the gel, which is an advantage over previous methods. DNA less than 50 kilobases can be separated without distortion even at high voltage with a nonalternating contour-clamped homogeneous field. Decreased band broadening in DNA less than 200 bases can be achieved with a contour-clamped inhomogeneous field.

The human genome is thought to harbor 50,000 to 100,000 genes, of which about half have been sampled to date in the form of expressed sequence tags. An international consortium was organized to develop and map gene-based sequence tagged site markers on a set of two radiation hybrid panels and a yeast artificial chromosome library. More than 16,000 human genes have been mapped relative to a framework map that contains about 1000 polymorphic genetic markers. The gene map unifies the existing genetic and physical maps with the nucleotide and protein sequence databases in a fashion that should speed the discovery of genes underlying inherited human disease. The integrated resource is available through a site on the World Wide Web at http://www.ncbi.nlm.nih.gov/SCIENCE96/.

Y chromosome haplotypes are particularly useful in deciphering human evolutionary history because they accentuate the effects of drift, migration, and range expansion. Significant acceleration of Y biallelic marker discovery and subsequent typing involving heteroduplex detection has been achieved by implementing an innovative and cost-efficient method called denaturing high-performance liquid chromatography (DHPLC). The power of the method resides in its sensitivity and ability to rapidly compare amplified sequences in an automated manner. We have determined the allelic states of 22 Y polymorphisms; 19 of which are unreported, in 718 diverse extant chromosomes; established haplotype frequencies; and deduced a phylogeny. All major geographic regions, including Eurasia, are characterized by mutations reflecting episodes of genetic drift and expansion. Most biallelic markers are localized regionally. However, some show wider dispersal and designate older, core haplotypes. One transversion defines a major haplogroup that distinguishes a previously unknown deep, apparently non-African branch. It provides evidence of an ancient bottleneck event. It is now possible to anticipate the inevitable detailed reconstruction of human Y chromosome genealogy based on several tens to even hundreds of these important polymorphisms.

A deletion map of the human Y chromosome was constructed by testing 96 individuals with partial Y chromosomes for the presence or absence of many DNA loci. The individuals studied included XX males, XY females, and persons in whom chromosome banding had revealed translocated, deleted, isodicentric, or ring Y chromosomes. Most of the 132 Y chromosomal loci mapped were sequence-tagged sites, detected by means of the polymerase chain reaction. These studies resolved the euchromatic region (short arm, centromere, and proximal long arm) of the Y chromosome into 43 ordered intervals, all defined by naturally occurring chromosomal breakpoints and averaging less than 800 kilobases in length. This deletion map should be useful in identifying Y chromosomal genes, in exploring the origin of chromosomal disorders, and in tracing the evolution of the Y chromosome.

The human genome is thought to harbor 50,000 to 100,000 genes, of which about half have been sampled to date in the form of expressed sequence tags. An international consortium was organized to develop and map gene-based sequence tagged site markers on a set of two radiation hybrid panels and a yeast artificial chromosome library. More than 16,000 human genes have been mapped relative to a framework map that contains about 1000 polymorphic genetic markers. The gene map unifies the existing genetic and physical maps with the nucleotide and protein sequence databases in a fashion that should speed the discovery of genes underlying inherited human disease. The integrated resource is available through a site on the World Wide Web at http://www.ncbi.nlm.nih.gov/SCIENCE96/.

The human Y chromosome was physically mapped by assembling 196 recombinant DNA clones, each containing a segment of the chromosome, into a single overlapping array. This array included more than 98 percent of the euchromatic portion of the Y chromosome. First, a library of yeast artificial chromosome (YAC) clones was prepared from the genomic DNA of a human XYYYY male. The library was screened to identify clones containing 160 sequence-tagged sites and the map was then constructed from this information. In all, 207 Y-chromosomal DNA loci were assigned to 127 ordered intervals on the basis of their presence or absence in the YAC's, yielding ordered landmarks at an average spacing of 220 kilobases across the euchromatic region. The map reveals that Y-chromosomal genes are scattered among a patchwork of X-homologous, Y-specific repetitive, and single-copy DNA sequences. This map of overlapping clones and ordered, densely spaced markers should accelerate studies of the chromosome.

Excellent resolution of chromosomal DNA molecules from Saccharomyces cerevisiae, Candida albicans and Schizosaccharomyces pombe has been obtained using alternating contour-clamped homogeneous electric field (CHEF) gel electrophoresis. The largest of these molecules is greater than 5 Mb in size and is resolved after 130 hours in a 0.6% agarose gel at a field strength of 1.3 V/cm and a switching interval of 1 hour. Separation of concatamers of phage lambda DNA reveals four regions of resolution in alternating CHEF gel electrophoresis. There are two regions of good resolution in which mobility approximates a linear function of molecular weight. These are separated by a region of lower resolution and bounded at high molecular weights by a region of little or no resolution. The four regions are of practical and possibly theoretical importance.

Optic nerve degeneration caused by glaucoma is a leading cause of blindness worldwide. Patients affected by the normal-pressure form of glaucoma are more likely to harbor risk alleles for glaucoma-related optic nerve disease. We have performed a meta-analysis of two independent genome-wide association studies for primary open angle glaucoma (POAG) followed by a normal-pressure glaucoma (NPG, defined by intraocular pressure (IOP) less than 22 mmHg) subgroup analysis. The single-nucleotide polymorphisms that showed the most significant associations were tested for association with a second form of glaucoma, exfoliation-syndrome glaucoma. The overall meta-analysis of the GLAUGEN and NEIGHBOR dataset results (3,146 cases and 3,487 controls) identified significant associations between two loci and POAG: the CDKN2BAS region on 9p21 (rs2157719 [G], OR = 0.69 [95%CI 0.63-0.75], p = 1.86×10⁻¹⁸), and the SIX1/SIX6 region on chromosome 14q23 (rs10483727 [A], OR = 1.32 [95%CI 1.21-1.43], p = 3.87×10⁻¹¹). In sub-group analysis two loci were significantly associated with NPG: 9p21 containing the CDKN2BAS gene (rs2157719 [G], OR = 0.58 [95% CI 0.50-0.67], p = 1.17×10⁻¹²) and a probable regulatory region on 8q22 (rs284489 [G], OR = 0.62 [95% CI 0.53-0.72], p = 8.88×10⁻¹⁰). Both NPG loci were also nominally associated with a second type of glaucoma, exfoliation syndrome glaucoma (rs2157719 [G], OR = 0.59 [95% CI 0.41-0.87], p = 0.004 and rs284489 [G], OR = 0.76 [95% CI 0.54-1.06], p = 0.021), suggesting that these loci might contribute more generally to optic nerve degeneration in glaucoma. Because both loci influence transforming growth factor beta (TGF-beta) signaling, we performed a genomic pathway analysis that showed an association between the TGF-beta pathway and NPG (permuted p = 0.009). These results suggest that neuro-protective therapies targeting TGF-beta signaling could be effective for multiple forms of glaucoma.

Retinal pigment epithelial (RPE) cell dysfunction plays a central role in various retinaldegenerative diseases, but knowledge is limited regarding the pathways responsible for adult RPE stress responses in vivo.RPE mitochondrial dysfunction has been implicated in the pathogenesis of several forms of retinal degeneration.Here we have shown that postnatal ablation of RPE mitochondrial oxidative phosphorylation in mice triggers gradual epithelium dedifferentiation, typified by reduction of RPE-characteristic proteins and cellular hypertrophy.The electrical response of the retina to light decreased and photoreceptors eventually degenerated.Abnormal RPE cell behavior was associated with increased glycolysis and activation of, and dependence upon, the hepatocyte growth factor/met proto-oncogene pathway.RPE dedifferentiation and hypertrophy arose through stimulation of the AKT/mammalian target of rapamycin (AKT/mTOR) pathway.Administration of an oxidant to wild-type mice also caused RPE dedifferentiation and mTOR activation.Importantly, treatment with the mTOR inhibitor rapamycin blunted key aspects of dedifferentiation and preserved photoreceptor function for both insults.These results reveal an in vivo response of the mature RPE to diverse stressors that prolongs RPE cell survival at the expense of epithelial attributes and photoreceptor function.Our findings provide a rationale for mTOR pathway inhibition as a therapeutic strategy for retinal degenerative diseases involving RPE stress.

Primary open-angle glaucoma (POAG) is a leading cause of blindness worldwide. To identify new susceptibility loci, we performed meta-analysis on genome-wide association study (GWAS) results from eight independent studies from the United States (3,853 cases and 33,480 controls) and investigated the most significantly associated SNPs in two Australian studies (1,252 cases and 2,592 controls), three European studies (875 cases and 4,107 controls) and a Singaporean Chinese study (1,037 cases and 2,543 controls). A meta-analysis of the top SNPs identified three new associated loci: rs35934224[T] in TXNRD2 (odds ratio (OR) = 0.78, P = 4.05 × 10(-11)) encoding a mitochondrial protein required for redox homeostasis; rs7137828[T] in ATXN2 (OR = 1.17, P = 8.73 × 10(-10)); and rs2745572[A] upstream of FOXC1 (OR = 1.17, P = 1.76 × 10(-10)). Using RT-PCR and immunohistochemistry, we show TXNRD2 and ATXN2 expression in retinal ganglion cells and the optic nerve head. These results identify new pathways underlying POAG susceptibility and suggest new targets for preventative therapies.

Primary open-angle glaucoma (POAG), is a heritable common cause of blindness world-wide. To identify risk loci, we conduct a large multi-ethnic meta-analysis of genome-wide association studies on a total of 34,179 cases and 349,321 controls, identifying 44 previously unreported risk loci and confirming 83 loci that were previously known. The majority of loci have broadly consistent effects across European, Asian and African ancestries. Cross-ancestry data improve fine-mapping of causal variants for several loci. Integration of multiple lines of genetic evidence support the functional relevance of the identified POAG risk loci and highlight potential contributions of several genes to POAG pathogenesis, including SVEP1, RERE, VCAM1, ZNF638, CLIC5, SLC2A12, YAP1, MXRA5, and SMAD6. Several drug compounds targeting POAG risk genes may be potential glaucoma therapeutic candidates.

The Royal College of Surgeons (RCS) rat is a widely studied animal model of retinal degeneration in which the inability of the retinal pigment epithelium (RPE) to phagocytize shed photoreceptor outer segments leads to a progressive loss of rod and cone photoreceptors. We recently used positional cloning to demonstrate that the gene Mertk likely corresponds to the retinal dystrophy (rdy) locus of the RCS rat. In the present study, we sought to determine whether gene transfer of Mertk to a RCS rat retina would result in correction of the RPE phagocytosis defect and preservation of photoreceptors. We used subretinal injection of a recombinant replication-deficient adenovirus encoding rat Mertk to deliver the gene to the eyes of young RCS rats. Electrophysiological assessment of animals 30 days after injection revealed an increased sensitivity of treated eyes to low-intensity light. Histologic and ultrastructural assessment demonstrated substantial sparing of photoreceptors, preservation of outer segment structure, and correction of the RPE phagocytosis defect in areas surrounding the injection site. Our results provide definitive evidence that mutation of Mertk underlies the RCS retinal dystrophy phenotype, and that the phenotype can be corrected by treatment of juvenile animals. To our knowledge, this is the first demonstration of complementation of both a functional cellular defect (phagocytosis) and a photoreceptor degeneration by gene transfer to the RPE. These results, together with the recent discovery of MERTK mutations in individuals with retinitis pigmentosa, emphasize the importance of the RCS rat as a model for gene therapy of diseases that arise from RPE dysfunction.

A molecular karyotype of Neurospora crassa was obtained by using an alternating-field gel electrophoresis system which employs contour-clamped homogeneous electric fields. The migration of all seven N. crassa chromosomal DNAs was defined, and five of the seven molecules were separated from one another. The estimated sizes of these molecules, based on their migration relative to Schizosaccharomyces pombe chromosomal DNA molecules, are 4 to 12.6 megabases. The seven linkage groups were correlated with specific chromosomal DNA bands by hybridizing transfers of contour-clamped homogeneous electric field gels with radioactive probes specific to each linkage group. The mobilities of minichromosomal DNAs generated from translocation strains were also examined. The methods used for preparation of chromosomal DNA molecules and the conditions for their separation should be applicable to other filamentous fungi.

To determine whether mice that are homozygous for a targeted disruption of the Mer receptor tyrosine kinase gene (mer(kd)) manifest a retinal dystrophy phenotype similar to RCS rats, which carry a mutation in the orthologous gene MERTK:Eyes of mer(kd) and C57BL/6 wild-type (WT) mice were examined by light and electron microscopy, whole-eye rhodopsin measurement, and Ganzfeld electroretinography (ERG).The mer(kd) mice showed rapid, progressive degeneration of the photoreceptors (PRs). Features of the phenotype common to mer(kd) mice and RCS rats included the absence or near absence of phagosomes in the retinal pigment epithelium (RPE) at the peak of outer segment (OS) disc shedding, accumulation of debris and whorls of membranes at the RPE-OS interface, transient supernormal rhodopsin content and OS lengths, the presence of OS vacuoles beginning at early ages, and a relatively slow removal of pyknotic PR nuclei. Most PRs were missing, and OS debris was removed by approximately postnatal day (P)45. Scotopic ERG responses were lower than age-matched WT responses and declined with PR loss. Photopic responses were preserved better than scotopic responses, corresponding with preferential cone preservation as judged histologically. ERG amplitudes were usually unmeasurable beyond P40, although a small-amplitude scotopic threshold response (STR) could still be elicited at P253 in some mice when only scattered PR nuclei remained.Ablation of Mer function in mer(kd) mice results in a retinal phenotype almost identical with that of RCS rats. The similarity in phenotypes between the two rodent models suggests that an RPE phagocytic defect is a feature of all types of retinal degeneration caused by loss of function of Mer tyrosine kinase, perhaps including mutations in human MERTK.

The RCS rat is a widely studied model of recessively inherited retinal degeneration. The genetic defect, known as rdy (retinal dystrophy), results in failure of the retinal pigment epithelium (RPE) to phagocytize shed photoreceptor outer segment membranes. We previously used positional cloning andin vivo genetic complementation to demonstrate thatMertk is the gene for rdy. We have now used a rat primary RPE cell culture system to demonstrate that the RPE is the site of action of Mertk and to obtain functional evidence for a key role of Mertk in RPE phagocytosis. We found that Mertk protein is absent from RCS, but not wild-type, tissues and cultured RPE cells. Delivery of rat Mertk to cultured RCS RPE cells by means of a recombinant adenovirus restored the cells to complete phagocytic competency. Infected RCS RPE cells ingested exogenous outer segments to the same extent as wild-type RPE cells, but outer segment binding was unaffected. Mertk protein progressively co-localized with outer segment material during phagocytosis by primary RPE cells, and activated Mertk accumulated during the early stages of phagocytosis by RPE-J cells. We conclude that Mertk likely functions directly in the RPE phagocytic process as a signaling molecule triggering outer segment ingestion. The RCS rat is a widely studied model of recessively inherited retinal degeneration. The genetic defect, known as rdy (retinal dystrophy), results in failure of the retinal pigment epithelium (RPE) to phagocytize shed photoreceptor outer segment membranes. We previously used positional cloning andin vivo genetic complementation to demonstrate thatMertk is the gene for rdy. We have now used a rat primary RPE cell culture system to demonstrate that the RPE is the site of action of Mertk and to obtain functional evidence for a key role of Mertk in RPE phagocytosis. We found that Mertk protein is absent from RCS, but not wild-type, tissues and cultured RPE cells. Delivery of rat Mertk to cultured RCS RPE cells by means of a recombinant adenovirus restored the cells to complete phagocytic competency. Infected RCS RPE cells ingested exogenous outer segments to the same extent as wild-type RPE cells, but outer segment binding was unaffected. Mertk protein progressively co-localized with outer segment material during phagocytosis by primary RPE cells, and activated Mertk accumulated during the early stages of phagocytosis by RPE-J cells. We conclude that Mertk likely functions directly in the RPE phagocytic process as a signaling molecule triggering outer segment ingestion. Phagocytosis is a process by which large particles are internalized by cells to form phagosomes. The process can be divided into three phases: binding, ingestion, and digestion. Retinal pigment epithelial (RPE) 1The abbreviations used are: RPEretinal pigment epitheliumOSouter segmentPpostnatal daym.o.i.multiplicity of infectionWGAwheat germ agglutininDMEMDulbecco's modified Eagle's mediumEndo Hendo-β-N-acetylglucosaminidase HPNGase Fpeptide N-glycosidase FGFPgreen fluorescent protein1The abbreviations used are: RPEretinal pigment epitheliumOSouter segmentPpostnatal daym.o.i.multiplicity of infectionWGAwheat germ agglutininDMEMDulbecco's modified Eagle's mediumEndo Hendo-β-N-acetylglucosaminidase HPNGase Fpeptide N-glycosidase FGFPgreen fluorescent protein cells, which form a polarized epithelium between the photoreceptor cells and the choroid in the outer retina, phagocytize more biomass than any other mammalian cell type (1.Bok D. Young R.W. Zinn K.E. Marmor M.F. The Retinal Pigment Epithelium. Harvard University Press, Cambridge, MA1979: 148-174Google Scholar). The RPE phagocytizes photoreceptor outer segment (OS) membranes (2.Young R.W. Bok D. J. Cell Biol. 1969; 42: 392-403Crossref PubMed Scopus (900) Google Scholar) that are shed as part of the normal ongoing process of photoreceptor OS renewal (3.Young R.W. Investig. Ophthalmol. Vis. Sci. 1978; 17: 105-116PubMed Google Scholar). Failure of OS membrane uptake leads to photoreceptor cell death (4.Bok D. Hall M.O. J. Cell Biol. 1971; 49: 664-682Crossref PubMed Scopus (438) Google Scholar), as illustrated by the RCS rat, a widely used model for recessively inherited retinal degeneration. The RCS mutation rdy (retinal dystrophy) causes, either directly or indirectly, a defect in RPE phagocytosis (4.Bok D. Hall M.O. J. Cell Biol. 1971; 49: 664-682Crossref PubMed Scopus (438) Google Scholar). This defect leads to an accumulation of shed OS membranes in the subretinal space (4.Bok D. Hall M.O. J. Cell Biol. 1971; 49: 664-682Crossref PubMed Scopus (438) Google Scholar) and a rapid and progressive degeneration of photoreceptor cells (5.Dowling J.E. Sidman R.L. J. Cell Biol. 1962; 14: 73-109Crossref PubMed Scopus (596) Google Scholar). retinal pigment epithelium outer segment postnatal day multiplicity of infection wheat germ agglutinin Dulbecco's modified Eagle's medium endo-β-N-acetylglucosaminidase H peptide N-glycosidase F green fluorescent protein retinal pigment epithelium outer segment postnatal day multiplicity of infection wheat germ agglutinin Dulbecco's modified Eagle's medium endo-β-N-acetylglucosaminidase H peptide N-glycosidase F green fluorescent protein The molecular mechanisms of RPE phagocytosis are unclear. Studies of the internalization of exogenous OS by cultured primary RPE cells suggested a receptor-mediated process (6.Mayerson P.L. Hall M.O. J. Cell Biol. 1986; 103: 299-308Crossref PubMed Scopus (113) Google Scholar, 7.Hall M.O. Abrams T. Exp. Eye Res. 1987; 45: 907-922Crossref PubMed Scopus (82) Google Scholar, 8.Laird D.W. Molday R.S. Investig. Ophthalmol. Vis. Sci. 1988; 29: 419-428PubMed Google Scholar). Inhibition of the RPE cell culture phagocytic assay by anti-receptor antibodies or competitive ligands suggested several specific proteins that might play a role in the process, including the mannose receptor (9.Boyle D. Tien L.F. Cooper N.G. Shepherd V. McLaughlin B.J. Investig. Ophthalmol. Vis. Sci. 1991; 32: 1464-1470PubMed Google Scholar, 10.Shepherd V.L. Tarnowski B.I. McLaughlin B.J. Investig. Ophthalmol. Vis. Sci. 1991; 32: 1779-1784PubMed Google Scholar), CD36 (11.Ryeom S.W. Sparrow J.R. Silverstein R.L. J. Cell Sci. 1996; 109: 387-395Crossref PubMed Google Scholar), and αvβ5 integrin (12.Finnemann S.C. Bonilha V.L. Marmorstein A.D. Rodriguez-Boulan E. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12932-12937Crossref PubMed Scopus (309) Google Scholar, 13.Miceli M.V. Newsome D.A. Tate Jr., D.J. Investig. Ophthalmol. Vis. Sci. 1997; 38: 1588-1597PubMed Google Scholar, 14.Lin H. Clegg D.O. Investig. Ophthalmol. Vis. Sci. 1998; 39: 1703-1712PubMed Google Scholar). Inhibition of αvβ5 integrin function disrupts the OS binding phase of RPE phagocytosis, whereas the mannose receptor and CD36 have been implicated in both OS binding and ingestion. Cultured RCS RPE cells bind exogenous OS at wild-type levels. However, only a small percentage of bound OS are ingested by RCS RPE cells (15.Chaitin M.H. Hall M.O. Investig. Ophthalmol. Vis. Sci. 1983; 24: 812-820PubMed Google Scholar), indicating that the protein encoded by therdy locus is critical, directly or indirectly, for OS uptake. The gene corresponding to rdy remained unknown until recently. The mannose receptor protein and messenger RNA are present in the RPE of both wild-type and RCS rats from postnatal day (P) 5 to adult (16.Wilt S.D. Greaton C.J. Lutz D.A. McLaughlin B.J. Exp. Eye Res. 1999; 69: 405-411Crossref PubMed Scopus (12) Google Scholar). CD36 null mice have been reported to have normal electroretinography and retinal histology (17.Silverstein R.L. Sparrow J.R. Ryeom S.W. Exp. Eye Res. 1998; 67: 534Google Scholar). These data suggest that neither the mannose receptor nor CD36 is the gene mutated in the RCS rat. We used positional cloning to identify a mutation in the receptor tyrosine kinase gene Mertk in the RCS rat. A deletion of RCS genomic DNA results in expression of an aberrant Mertktranscript with a translation termination signal after codon 20 (18), likely a complete loss-of-function, or null, allele. Mertkwas an appropriate candidate for rdy in light of evidence that a signaling defect might underlie the RCS RPE phagocytic phenotype (19.Chaitin M.H. Hall M.O. Investig. Ophthalmol. Vis. Sci. 1983; 24: 821-831PubMed Google Scholar, 20.Heth C.A. Schmidt S.Y. Investig. Ophthalmol. Vis. Sci. 1992; 33: 2839-2847PubMed Google Scholar, 21.Heth C.A. Marescalchi P.A. Investig. Ophthalmol. Vis. Sci. 1994; 35: 409-416PubMed Google Scholar). The discovery of mutations in the human ortholog,MERTK, in individuals with retinitis pigmentosa indicated that Mertk is essential for maintenance of the mammalian retina (22.Gal A. Li Y. Thompson D.A. Weir J. Orth U. Jacobson S.G. Apfelstedt-Sylla E. Vollrath D. Nat. Genet. 2000; 26: 270-271Crossref PubMed Scopus (533) Google Scholar). Subsequently, in vivo genetic complementation of the RCS phenotype by viral mediated gene transfer conclusively demonstrated that Mertk is the gene for rdy(23.Vollrath D. Feng W. Duncan J. Yasumura D. D'Cruz P.M. Chappelow A. Matthes M.T. Kay M.A. LaVail M.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 12584-12589Crossref PubMed Scopus (243) Google Scholar). The identification of Mertk provides an initial focus for elucidating molecular mechanisms of RPE phagocytosis. In the present study, we sought to determine whether the site of action ofMertk was indeed the RPE, as suggested by genetic chimera experiments (24.Mullen R.J. LaVail M.M. Science. 1976; 192: 799-801Crossref PubMed Scopus (421) Google Scholar) and, if so, whether Mertk protein was directly involved in the ingestion step of OS phagocytosis. We tested whether viral mediated gene transfer of Mertk to cultured RCS RPE cells could complement their ingestion defect. We also generated a polyclonal antibody directed against rat Mertk and used it to examine the activation state and subcellular localization of the receptor over a time course of OS phagocytosis by cultured RPE cells. All tissue culture reagents were purchased from Invitrogen. Primary RPE cells were isolated essentially as described (25.Chang C.W. Roque R.S. Defoe D.M. Caldwell R.B. Curr. Eye Res. 1991; 10: 1081-1086Crossref PubMed Scopus (78) Google Scholar) from pigmented RCS-p + and wild-type congenic RCS-rdy+p+ rats at P6 to P9. The RPE cells were plated on 4-well Lab-Tek chamber slides (Nunc, Naperville, IL) for phagocytic assays or on cell culture dishes (Corning Glass) for protein studies. The cultures were maintained in low glucose Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum and used after the cells grew confluent. OS were isolated from P30 Sprague-Dawley rats (Simonsen Laboratories, Gilroy, CA) as described (15.Chaitin M.H. Hall M.O. Investig. Ophthalmol. Vis. Sci. 1983; 24: 812-820PubMed Google Scholar), and the purified OS were stored in 1× BSS (10 mm HEPES, 137 mm NaCl, 5.36 mm KCl, 0.34 mm Na2HPO4, 0.44 mm KH2PO4, 0.81 mmMgSO4, 1.27 mm CaCl2, pH 7.4) containing 5% sucrose at −80 °C. All animal procedures adhered to the Association for Research in Vision and Ophthalmology Resolution on the Use of Animals in Research. The rat RPE cell line RPE-J and the rat kidney fibroblast cell line NRK-49F were obtained from American Type Culture Collection. RPE-J cells were cultured as described (26.Nabi I.R. Mathews A.P. Cohen-Gould L. Gundersen D. Rodriguez-Boulan E. J. Cell Sci. 1993; 104: 37-49PubMed Google Scholar), and NRK-49F cells were maintained as recommended by the company. A DNA fragment encoding the 103 C-terminal amino acids of rat Mertk was inserted into pGEX-1 (AMRAD Corp. Ltd., Australia) and transformed into bacterial strain BL21(DE3)pLysS (Invitrogen, Carlsbad, CA) to produce a glutathione S-transferase fusion protein. The fusion protein was purified from crude bacterial lysate by affinity chromatography on immobilized glutathione (Sigma) and injected into rabbits to generate polyclonal antibodies. A recombinant adenovirus containing the complete open reading frame of rat Mertk driven by a cytomegalovirus promoter was constructed as described (23.Vollrath D. Feng W. Duncan J. Yasumura D. D'Cruz P.M. Chappelow A. Matthes M.T. Kay M.A. LaVail M.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 12584-12589Crossref PubMed Scopus (243) Google Scholar). To deliver Mertk into RPE cells, Ad-Mertk was added to confluent RPE cultures at various multiplicities of infection (m.o.i.) and incubated for 42 h at 37 °C. A recombinant Ad-GFP (a gift from Dr. Yongjian Wu, Stanford University) was used as a control. RPE-J cells were grown on Matrigel (Fisher) for 6 days and then infected with Ad-Mertkat an m.o.i. of 2 for 42 h at 32 °C. NRK-49F cells were incubated with Ad-Mertk or Ad-GFP at an m.o.i. of 10 for 42 h at 37 °C. Wheat germ agglutinin (WGA)-enriched samples, deglycosylated samples, immunoprecipitation samples, and whole cell lysates were separated by 6 or 7.5% SDS-PAGE and transferred to a nitrocellulose membrane (Fisher). Mertk protein was detected by the C-terminal polyclonal antibody (1:1,000 dilution) followed by horseradish peroxidase-conjugated secondary antibody and the Super Signal West Pico Chemiluminescent Substrate system (Pierce). Pre-immune serum was used as a negative control. A polyclonal antibody directed against the ectodomain of murine Mertk (R & D Systems, Minneapolis, MN), which cross-reacts weakly with rat Mertk, was also used for immunoblotting (1:100 dilution). Tyrosine-phosphorylated Mertk was detected in immunoprecipitation samples by immunoblot with an anti-phosphotyrosine monoclonal antibody (P-Tyr-100, Cell Signaling, Beverly, MA). Neural retina, RPE/sclera, brain, and kidney were dissected from RCS-p+ and wild-type RCS-rdy+p+ rats at P23–P26 and homogenized in 1% Nonidet P-40 lysis buffer (50 mm Tris-HCl, 150 mm NaCl, 2 mmEDTA, 1% Nonidet P-40, pH 8.0) containing a protease inhibitor mixture (Roche Molecular Biochemicals). Equivalent growth areas of wild-type and mutant rat primary RPE cultures were washed three times with Dulbecco's phosphate-buffered saline and treated with 1% Nonidet P-40 lysis buffer on ice for 20 min before cell lysates were collected. Tissue homogenates and cell lysates were centrifuged at 14,000 rpm in a Sorvall Microspin 24S centrifuge for 15 min at 4 °C. Supernatants were collected and incubated with WGA-Sepharose beads (Sigma) for 3 h at 4 °C with rotation. Beads were washed five times with 1% Nonidet P-40 lysis buffer, and glycoproteins were dissociated from the beads by boiling for 5 min in 2× loading buffer (0.125m Tris-HCl, 4% SDS, 20% glycerol, 0.2 mdithiothreitol, 0.02% bromphenol blue, pH 6.8). Glycoproteins enriched with WGA beads were dissociated from the beads in 1× denaturing buffer (New England Biolabs, Beverly, MA) and treated according to the manufacturer's instructions with either PNGase F (New England Biolabs), to remove all forms of the N-linked oligosaccharides, or with Endo H (New England Biolabs), to cleave the chitobiose core of high mannose and hybrid form of N-linked oligosaccharides. Confluent wild-type primary RPE cells were washed with DMEM and fixed in 4% paraformaldehyde for 10 min at room temperature. Cell membranes were permeabilized with 0.5% Triton X-100, and cells were incubated for 30 min with fluorescein phalloidin (Molecular Probes, Eugene, OR) to detect F-actin by fluorescence microscopy. The ability of RPE cells to phagocytize OS was measured as reported previously (27.Feng W. Lutz D.A. McLaughlin B.J. Investig. Ophthalmol. Vis. Sci. 1996; 37: 378Google Scholar). OS were suspended in DMEM containing 5% fetal bovine serum and 5% sucrose at a concentration of 1 × 107 OS per ml. One ml of OS was added to each well of a 4-well chamber slide and incubated with adenovirus-infected or uninfected cells for 4 h at 37 °C. Then unbound OS were washed away with DMEM, and cells were fixed for 15 min with 4% paraformaldehyde in phosphate-buffered saline (137 mm NaCl, 3 mm KCl, 5 mm Na2HPO4, 2 mm,KH2PO4, pH 7.4). To distinguish total and bound OS, samples were divided into two groups. Each group contained 2 wells of cells. Group 1 was permeabilized with 0.5% Triton X-100 and group 2 remained unpermeabilized. OS were immunolabeled with anti-rhodopsin monoclonal antibody Rho 4D2 (8.Laird D.W. Molday R.S. Investig. Ophthalmol. Vis. Sci. 1988; 29: 419-428PubMed Google Scholar) (kindly provided by Dr. Robert S. Molday), followed by a Texas Red-conjugated anti-mouse IgG (Molecular Probes). Fluorescent labeling was observed under a Zeiss fluorescence microscope, and images were taken from 10–15 random fields (0.036 mm2 per field) for each group. OS with an estimated diameter of 1 μm or larger were manually counted. The total number of OS (bound plus ingested) was obtained from group 1 samples, and the number of bound OS was obtained from group 2 samples. The number of ingested OS was obtained by subtracting bound OS from total OS. For each experimental condition, the assay was repeated at least three times. Results for each condition were presented as a means ± S.E. A Student's t test was used for statistical evaluation. Confluent wild-type primary RPE cells were incubated with OS for 1–3 h, and unbound OS were removed by washing with DMEM. Cells were fixed in 100% ethanol for 5 min at −20 °C. Localization of the remaining OS and Mertk was examined by immunolabeling with the Rho 4D2 (1:100) and the C-terminal antibody (1:100) followed by Texas Red-conjugated anti-mouse IgG and Oregon Green-conjugated anti-rabbit IgG (Molecular Probes). RPE-J cells were incubated with OS (3 × 107 per ml) or control medium for 1–3 h at 32 °C. The cells were washed twice with cold phosphate-buffered saline containing 1 mm sodium orthovanadate (Sigma) and lysed in RIPA buffer (50 mm Tris, 150 mm NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, pH 8.0) containing 10 mm sodium orthovanadate and the protease inhibitor mixture. The cell lysate was centrifuged at 16,000 ×g for 10 min at 4 °C, and the supernatant was incubated with 20 μl of the Mertk C-terminal antiserum overnight at 4 °C. The antigen-antibody complex was precipitated by protein A-Sepharose beads (Amersham Biosciences AB), and proteins were dissociated from the beads by boiling for 5 min in 2× loading buffer. Our molecular genetic data predict that only a 20-amino acid N-terminal peptide of Mertk can be synthesized in the RCS rat (18.D'Cruz P.M. Yasumura D. Weir J. Matthes M.T. Abderrahim H. LaVail M.M. Vollrath D. Hum. Mol. Genet. 2000; 9: 645-652Crossref PubMed Scopus (710) Google Scholar). It is therefore likely that most or all of the protein is missing from RCS tissues. To test this prediction, we generated a polyclonal antibody directed against the C terminus of rat Mertk and assessed its specificity by immunoblotting. As shown in Fig. 1 A, anti-Mertk recognized two bands of ∼200 and 150 kDa from rat kidney fibroblasts infected with a recombinant adenovirus, Ad-Mertk, which expresses ratMertk (23.Vollrath D. Feng W. Duncan J. Yasumura D. D'Cruz P.M. Chappelow A. Matthes M.T. Kay M.A. LaVail M.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 12584-12589Crossref PubMed Scopus (243) Google Scholar). These bands were absent from Ad-GFP-infected or uninfected cells, indicating that the antibody specifically recognizes Mertk. Pre-immune serum did not detect bands in any of the samples (data not shown). The C-terminal antibody was used to assess Mertk expression in primary RPE cultures from RCS or a wild-type, congenic strain, RCS-rdy +. As expected, anti-Mertk recognized a single band of about 200 kDa from wild-type RPE cells but not from RCS RPE cells (Fig. 1 B). We next used the C-terminal antibody and a polyclonal antiserum that reacts with the Mertk ectodomain to examine various RCS and wild-type RCS-rdy +tissues including neural retina, RPE/sclera, brain, and kidney (Fig. 2). The C-terminal antibody (Fig. 2 A) recognized an ∼190-kDa band from each of the four wild-type tissues tested, as well as smaller bands from wild-type neural retina (∼160 kDa) and brain (∼150 kDa). None of these bands was present in any of the RCS samples. The ectodomain antibody (Fig. 2 B) recognized the same Mertk bands as the C-terminal antibody in wild-type tissues, although with lower avidity. The primary amino acid sequence of Mertk predicts a protein of about 107 kDa, significantly smaller than the proteins we detected. There are 14 putative N-linked glycosylation sites in the ectodomain of rat Mertk (GenBank™ accession number P57097), and evidence indicates that human MERTK from embryonic kidney cells is about 200 kDa in size (28.Chen J. Carey K. Godowski P.J. Oncogene. 1997; 14: 2033-2039Crossref PubMed Scopus (154) Google Scholar), consistent with our data. To determine whether the different protein bands we detected with anti-Mertk are due to differential glycosylation, WGA-enriched protein samples were digested with the deglycosylase, PNGase F. After PNGase F treatment, the 150–200-kDa bands disappeared, and a new band of about 120 kDa was detected by the C-terminal antibody (Fig. 3 A). The size of the new band is slightly larger than the predicted size of rat Mertk. These results indicate that rat Mertk exists in at least two glycosylated forms. To determine which of these forms are mature proteins, WGA-enriched proteins from brain were treated with Endo H. In the early steps of N-linked oligosaccharide processing in the endoplasmic reticulum and Golgi, oligosaccharides are Endo H-sensitive until Golgi mannosidase II removes the last two mannose residues from the final core of three residues present in a complex oligosaccharide (29.Alberts B. Bray D. Lewis J. Raff M. Roberts K. Watson J.D. Molecular Biology of the Cell. 3rd Ed. Garland Publishing, Inc., New York1994: 604-605Google Scholar). As shown in Fig. 3 B (lanes 1 and 2), the 190-kDa band remained intact after digestion with Endo H, indicating that the band corresponds to a mature form of Mertk. By contrast, Endo H treatment resulted in disappearance of the 150-kDa band and appearance of a smaller band (lane 2). The size of the new band was greater than that of the band produced by PNGase F treatment (compare lanes 2and 3). Extended Endo H treatment did not further reduce the size of the new band. The partial Endo H sensitivity of the 150-kDa protein indicates that it contains both complex and high mannose forms of N-linked oligosaccharides. After demonstrating that RCS RPE lacked Mertk protein, we sought to determine whether transfer of the gene to RCS RPE would complement the well described OS phagocytic defect in primary cell culture. Rat RPE cells in culture form a monolayer with polygonal cell morphology (Fig. 4 A). We chose Ad-Mertk as the means of delivering the gene to these cells because earlier studies had demonstrated that adenovirus efficiently infects primary RCS RPE (30.da Cruz L. Robertson T. Hall M.O. Constable I.J. Rakoczy P.E. Curr. Eye Res. 1998; 17: 668-672Crossref PubMed Google Scholar). As expected, when RCS RPE cells were incubated with Ad-GFP at a low m.o.i. of 2, almost all were found to be GFP-positive after 42 h (Fig. 4 B). Similarly, Mertk was delivered into RCS RPE cells with high efficiency by Ad-Mertk; cells infected at increasing m.o.i. values expressed proportionally more Mertk protein (Fig. 4 C). Even at an m.o.i. of 2, infected cells produced more Mertk than wild-type RPE. We assessed the effect of Mertk expression on the phagocytic ability of RCS RPE by a cell culture assay. RPE monolayers were incubated with purified rat OS for 4 h. The cells were then washed, fixed, and either permeabilized with Triton X-100 or not, and OS were detected with a monoclonal antibody directed against rhodopsin to determine the number of bound and ingested OS. Bound and ingested OS in RCS RPE, virus-infected RCS RPE, and wild-type RPE cells were quantified (Fig. 5 A). Consistent with the results of Chaitin and Hall (15.Chaitin M.H. Hall M.O. Investig. Ophthalmol. Vis. Sci. 1983; 24: 812-820PubMed Google Scholar), RCS RPE cells bound similar numbers of OS as did wild-type RPE (p = 0.70) but could not ingest any of the bound OS. By contrast, wild-type RPE cells ingested 73 ± 8% of total OS. Introduction of Mertk into RCS RPE dramatically altered the difference in OS ingestion between these two types of RPE cells (Fig. 5 A). Infection of RCS RPE by Ad-Mertk at an m.o.i. of 2 caused the infected cells to ingest as many OS as wild-type RPE (p = 0.84). The number of bound OS was not altered in comparison to uninfected RCS (p = 0.47) or wild-type RCS-rdy +cells (p = 0.70). The functional rescue that we observed is specifically due to Mertk because infection with Ad-GFP did not result in an increase in OS ingestion (p = 0.86). Additional Mertk did not increase OS ingestion in wild-type RPE (Fig. 5 A). Cells infected with Ad-Mertk at an m.o.i. of 2 ingested an equal amount of OS as compared with uninfected cells (p = 0.88), despite the higher level of Mertk in virus-infected cells (Fig. 5 B). The slight increase in the number of bound OS (Fig. 5 A) was not significant (p = 0.34). The preceding data demonstrate that the RPE is indeed the site of action of Mertk with respect to OS phagocytosis. To assess whether Mertk is directly involved in OS ingestion, we examined the subcellular localization of the protein during a time course of RPE phagocytosis. Double immunolabeling of uninfected wild-type RPE cells during the first 3 h of phagocytosis revealed progressive co-localization of Mertk with OS (Fig. 6). A small number of OS co-localized with punctate Mertk signals after 1 h of incubation (Fig. 6, A–C, arrowheads). The number OS with accompanying Mertk increased by 2 h (Fig. 6, D–F), and by 3 h, the patterns of OS staining and punctate Mertk staining were almost identical (Fig. 6, G–I). These data indicate that Mertk gradually accumulates around OS with a time course that is similar to the progressive ingestion of OS (7.Hall M.O. Abrams T. Exp. Eye Res. 1987; 45: 907-922Crossref PubMed Scopus (82) Google Scholar, 15.Chaitin M.H. Hall M.O. Investig. Ophthalmol. Vis. Sci. 1983; 24: 812-820PubMed Google Scholar). If Mertk is directly involved in OS ingestion, as suggested by the co-localization data, then the receptor should be increasingly activated after addition of OS. To test this hypothesis, we examined the activation state of the receptor by monitoring the extent of phosphorylated tyrosine residues in Mertk during the first 3 h of OS phagocytosis. To obtain a sufficient number of cells, we used RPE-J, a well characterized rat RPE cell line with the ability to phagocytize OS (12.Finnemann S.C. Bonilha V.L. Marmorstein A.D. Rodriguez-Boulan E. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12932-12937Crossref PubMed Scopus (309) Google Scholar, 26.Nabi I.R. Mathews A.P. Cohen-Gould L. Gundersen D. Rodriguez-Boulan E. J. Cell Sci. 1993; 104: 37-49PubMed Google Scholar), and infected them with Ad-Mertk (m.o.i. = 2) to enhance detection of tyrosine-phosphorylated receptor molecules. After 1 h of OS incubation, we did not detect activated Mertk by immunoprecipitation with anti-Mertk and immunoblotting with an anti-phosphotyrosine monoclonal antibody (Fig. 7). However, after 3 h, two tyrosine-phosphorylated forms of Mertk were readily detected (Fig. 7). Activated Mertk was not observed in the absence of added OS (Fig. 7). A parallel experiment with uninfected RPE-J cells yielded similar results, except the signals were much weaker (data not shown). We have transferred wild-type Mertk to RCS RPE cells, which we showed lacked Mertk protein, and completely corrected the phagocytic defect of the cells. These results definitively establish the RPE as the site of action of Mertk with respect to OS phagocytosis, as suggested previously (24.Mullen R.J. LaVail M.M. Science. 1976; 192: 799-801Crossref PubMed Scopus (421) Google Scholar). Moreover, the fact that Ad-Mertk-infected RCS cells bound and ingested OS at wild-type levels demonstrates that the rest of the phagocytic machinery in RCS RPE is normal and that RCS RPE cells have the same potential as wild-type RPE for OS internalization. Reported biochemical abnormalities of RCS RPE, such as increased calcium membrane conductance and altered cAMP and inositol phosphate second messenger metabolism (31.Strauss O. Stumpff F. Mergler S. Wienrich M. Wiederholt M. Acta Anat. 1998; 162: 101-111Crossref PubMed Scopus (75) Google Scholar), are likely secondary to the loss of Mertk function. Mertk did not affect the binding phase of phagocytosis in primary RPE cell culture; modest overexpression of the protein (m.o.i. = 2) in RCS RPE and wild-type RPE cells did not significantly increase OS binding. These results are consistent with previous reports indicating that αvβ5 integrin is a major OS binding receptor for RPE in cell culture (12.Finnemann S.C. Bonilha V.L. Marmorstein A.D. Rodriguez-Boulan E. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12932-12937Crossref PubMed Scopus (309) Google Scholar, 14.Lin H. Clegg D.O. Investig. Ophthalmol. Vis. Sci. 1998; 39: 1703-1712PubMed Google Scholar). However, αvβ5 integrin cannot be essential for retinal structure and function because mice with a targeted disruption of the β5 gene have normal retinal anatomy and electroretinography at 1 and 4 months of age, 2J. Duncan, X. Huang, M. M. LaVail, D. Sheppard, and D. Vollrath, unpublished observations. despite the fact that disc shedding and phagocytosis begin around P12. The apparent discrepancy between the role of αvβ5integrin in cell culture and in vivo may result from physical differences in the process of RPE phagocytosis in these two settings. In vivo, OS are closely apposed to RPE microvilli, whereas in cell culture, purified OS are suspended in culture medium and added to cells. Thus, OS binding in cell culture may not be relevant to, or may be substantially different from, the normalin vivo OS phagocytic process. By contrast, Mertk is required for RPE phagocytosis of OS both in vivo (23.Vollrath D. Feng W. Duncan J. Yasumura D. D'Cruz P.M. Chappelow A. Matthes M.T. Kay M.A. LaVail M.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 12584-12589Crossref PubMed Scopus (243) Google Scholar) and in cell culture (the present study). We generated a polyclonal antibody suitable for immunoblotting and demonstrated that the antibody specifically recognizes 190–200- and 150–160-kDa forms of Mertk in the RPE and assorted other tissues. These sizes are significantly larger than the molecular weight predicted on the basis of the rat primary amino acid sequence. We found that a large majority of the excess molecular weight is due to the presence of N-linked oligosaccharides and that the two forms arise from differential glycosylation. Both forms of the receptor present in RPE-J cells can be activated by OS (Fig. 7). It therefore appears that both forms are functional and probably localize to the plasma membrane. Mertk appears to be an integral component of the phagocytic machinery. During the early stages of RPE phagocytosis, Mertk progressively co-localized with OS. The time course of co-localization matched that of the activation of Mertk, as measured by tyrosine phosphorylation, suggesting that a close association with OS may be required to activate the receptor. Because Mertk only stimulated OS internalization and not binding, the protein must be critical for the ingestion phase. The delayed activation of Mertk is consistent with the observed initial delay in the kinetics of OS ingestion by cultured RPE cells (7.Hall M.O. Abrams T. Exp. Eye Res. 1987; 45: 907-922Crossref PubMed Scopus (82) Google Scholar, 12.Finnemann S.C. Bonilha V.L. Marmorstein A.D. Rodriguez-Boulan E. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12932-12937Crossref PubMed Scopus (309) Google Scholar,15.Chaitin M.H. Hall M.O. Investig. Ophthalmol. Vis. Sci. 1983; 24: 812-820PubMed Google Scholar). By 3 h of incubation, however, substantial OS ingestion has occurred (7.Hall M.O. Abrams T. Exp. Eye Res. 1987; 45: 907-922Crossref PubMed Scopus (82) Google Scholar), and about 70% of total OS are ingested by 4 h (Fig. 5 A). The fact that nearly all OS were accompanied by punctate Mertk signals at 3 h (Fig. 6) suggests that the receptor becomes internalized with OS as part of the phagosome. Further studies are required to address the turnover of Mertk. The requirement for Mertk in both RPE phagocytosis of OS in the RCS rat and macrophage phagocytosis of apoptotic cells in the Merkd mouse (32.Scott R.S. McMahon E.J. Pop S.M. Reap E.A. Caricchio R. Cohen P.L. Earp H.S. Matsushima G.K. Nature. 2001; 411: 207-211Crossref PubMed Scopus (907) Google Scholar), combined with the general similarities between RPE phagocytosis and the uptake of apoptotic cells by macrophages and other professional phagocytes, suggests that the two processes may share mechanistic features. Activation of Mertk could trigger an intracellular signaling pathway that controls rearrangement of cytoskeletal components necessary for OS or apoptotic cell ingestion. Phosphotyrosine accumulates within actin cups that form immediately beneath the site of apoptotic cell ingestion during macrophage phagocytosis (33.Leverrier Y. Ridley A.J. Curr. Biol. 2001; 11: 195-199Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Moreover, apoptotic cell uptake by professional and non-professional phagocytes requires a tyrosine kinase signaling pathway to activate CrkII and Rac (33.Leverrier Y. Ridley A.J. Curr. Biol. 2001; 11: 195-199Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 34.Albert M.L. Kim J.I. Birge R.B. Nat. Cell Biol. 2000; 2: 899-905Crossref PubMed Scopus (322) Google Scholar). TheCaenorhabditis elegans homologs CED-2 (CrkII) and CED-10 (Rac) are required for engulfment of apoptotic cells, demonstrating an ancient origin to at least part of this pathway (35.Reddien P.W. Horvitz H.R. Nat. Cell Biol. 2000; 2: 131-136Crossref PubMed Scopus (329) Google Scholar). This signaling pathway may also be activated during RPE phagocytosis of OS. It is not yet known whether phosphatidylserine plays a key role in the recognition of OS by RPE cells, as it does in the recognition of apoptotic cells by macrophages (36.Fadok V.A. Voelker D.R. Campbell P.A. Cohen J.J. Bratton D.L. Henson P.M. J. Immunol. 1992; 148: 2207-2216PubMed Google Scholar). It is interesting that annexin V binds avidly to purified rat OS, indicating that phosphatidylserine is exposed on the outside. 3W. Feng and D. Vollrath, unpublished observations. A secreted ligand of Mertk, Gas6 (28.Chen J. Carey K. Godowski P.J. Oncogene. 1997; 14: 2033-2039Crossref PubMed Scopus (154) Google Scholar, 37.Nagata K. Ohashi K. Nakano T. Arita H. Zong C. Hanafusa H. Mizuno K. J. Biol. Chem. 1996; 271: 30022-30027Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar), binds phosphatidylserine (38.Nakano T. Ishimoto Y. Kishino J. Umeda M. Inoue K. Nagata K. Ohashi K. Mizuno K. Arita H. J. Biol. Chem. 1997; 272: 29411-29414Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar) and may serve as a bridge between OS and Mertk at the RPE plasma membrane during phagocytosis, as we suggested previously (18.D'Cruz P.M. Yasumura D. Weir J. Matthes M.T. Abderrahim H. LaVail M.M. Vollrath D. Hum. Mol. Genet. 2000; 9: 645-652Crossref PubMed Scopus (710) Google Scholar). Consistent with this model, Hall and colleagues (39.Hall M.O. Prieto A.L. Obin M.S. Abrams T.A. Burgess B.L. Heeb M.J. Agnew B.J. Exp. Eye Res. 2001; 73: 509-520Crossref PubMed Scopus (72) Google Scholar) recently reported that Gas6 stimulates phagocytosis of exogenous OS by rat primary RPE cells. It will be of great interest to determine whether Gas6 plays a key role in RPE phagocytosis and/or internalization of apoptotic cellsin vivo. In summary, we have demonstrated that Mertk is an integral component of the RPE phagocytic process in cell culture, in which it probably functions to trigger ingestion of bound OS. Future studies on the interaction of Mertk with upstream and downstream proteins will help to elaborate the molecular mechanism of RPE phagocytosis. The common requirement for Mertk in uptake of apoptotic cells by professional phagocytes and OS phagocytosis by RPE indicates that elucidation of this mechanism may have general implications. We thank Jessica Weir for DNA sequencing and Nancy Lawson and Dean Cruz for help with the animals. We also thank Dr. Silvia Finnemann for detailed instructions for RPE-J culture.

To the Editor: Primary open-angle glaucoma (POAG) is an important cause of irreversible blindness worldwide (Quigley Quigley, 1996Quigley HA Number of people with glaucoma world-wide.Br J Ophthalmol. 1996; 80: 389-393Crossref PubMed Scopus (1885) Google Scholar). The disease results in a characteristic degeneration of the optic nerve that is usually associated with an elevation of intraocular pressure. Pressure within the eye is dependent on the rate of production of a fluid (aqueous humor) by the ciliary body and on the rate of removal of the fluid by the trabecular meshwork. Relatives of POAG patients have an increased risk of developing glaucoma, which suggests that genetic factors are an important component of POAG susceptibility (Leske Leske, 1983Leske MC The epidemiology of open-angle glaucoma: a review.Am J Epidemiol. 1983; 118: 166-191Crossref PubMed Scopus (411) Google Scholar). Adult-onset POAG is inherited as a non-Mendelian trait, whereas forms of juvenile-onset POAG exhibit autosomal-dominant inheritance (Wiggs et al. Wiggs et al., 1995Wiggs JL DelBono EA Schuman JS Hutchinson BT Walton DS Clinical features of five pedigrees genetically linked to the juvenile glaucoma locus on chromosome 1q21-q31.Ophthalmology. 1995; 102: 1782-1789Abstract Full Text PDF PubMed Scopus (66) Google Scholar). One locus for juvenile glaucoma was initially mapped to 1q23 (Sheffield et al. Sheffield et al., 1993Sheffield VC Stone EM Alward WLM Drack AV Johnson AT Streb LM Nichols BE Genetic linkage of familial open angle glaucoma to chromosome 1q21-q31.Nat Genet. 1993; 4: 47-50Crossref PubMed Scopus (389) Google Scholar; Richards et al. Richards et al., 1994Richards JE Lichter PR Boehnke M Uro JL Torrez D Wong D Johnson AT Mapping of a gene for autosomal dominant juvenile-onset open-angle glaucoma to chromosome 1q.Am J Hum Genet. 1994; 54: 62-70PubMed Google Scholar; Wiggs et al. Wiggs et al., 1994Wiggs JL Haines JL Paglinauan C Fine A Sporn C Lou D Genetic linkage of autosomal dominant juvenile glaucoma in 1q21-q31 in three affected pedigrees.Genomics. 1994; 21: 299-303Crossref PubMed Scopus (82) Google Scholar) and was subsequently refined to a 3-cM interval (Belmouden et al. Belmouden et al., 1997Belmouden A Adam MF de Dinechin SD Brézin AP Rigault P Chumakov I Bach JF et al.Recombinational and physical mapping of the locus for primary open-angle glaucoma (GLC1A) chromosome 1q23-q25.Genomics. 1997; 39: 348-358Crossref PubMed Scopus (26) Google Scholar). In recent studies, evaluation of candidate genes mapped to this region has led to the identification of mutations in the TIGR/Myocilin gene (Stone et al. Stone et al., 1997Stone EM Fingert JH Alward WLM Nguyen TD Polansky JR Sunden SLF Nishimura D et al.Identification of a gene that causes primary open angle glaucoma.Science. 1997; 275: 668-670Crossref PubMed Scopus (1191) Google Scholar). This gene was originally cloned, from cultured trabecular meshwork cells, as a steroid-response protein, named “trabecular meshwork-induced glucocorticoid response protein” (TIGR; Nguyen et al. Nguyen et al., 1993Nguyen TD Huang W Bloom E Polansky JR Glucocorticoid (GC) effects on HTM cells: molecular biology approaches.in: Lütjen-Drecoll E Basic aspects of glaucoma research III. Shattauer, Stuttgart1993: 331-343Google Scholar). The gene was isolated subsequently from a retinal cDNA library and was shown to be localized to the cilium connecting the inner and outer segments of photoreceptor cells (named “myocilin”; Kubota et al. Kubota et al., 1997Kubota R Noda S Wang Y Minoshima S Asakawa S Kudoh J Mashima Y et al.A novel myosin-like protein (myocilin) expressed in the connecting cilium of the photoreceptor: molecular cloning, tissue expression, and chromosomal mapping.Genomics. 1997; 41: 360-369Crossref PubMed Scopus (271) Google Scholar). Mutations have been detected in the TIGR/myocilin gene in juvenile- and adult-onset glaucoma pedigrees, and in populations of sporadic adult- and juvenile-onset patients (Adam et al. Adam et al., 1997Adam MF Belmouden A Binisti P Brézin AP Valtot F Béchetoille A Dascotte J-C et al.Recurrent mutations in a single exon encoding the evolutionarily conserved olfactomedin-homology domain of TIGR in familial open-angle glaucoma.Hum Mol Genet. 1997; 6: 2091-2097Crossref PubMed Scopus (247) Google Scholar; Stone et al. Stone et al., 1997Stone EM Fingert JH Alward WLM Nguyen TD Polansky JR Sunden SLF Nishimura D et al.Identification of a gene that causes primary open angle glaucoma.Science. 1997; 275: 668-670Crossref PubMed Scopus (1191) Google Scholar; Suzuki et al. Suzuki et al., 1997Suzuki Y Shirato S Taniguchi F Ohara K Nishimaki K Ohta S Mutations in the TIGR gene in familial primary open-angle glaucoma in Japan.Am J Hum Genet. 1997; 61: 1202-1204Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar; Alward et al. Alward et al., 1998Alward WLM Fingert JH Coote MA Johnson AT Lerner SF Junqua D Durcan FJ et al.Clinical features associated with mutations in the chromosome 1 open-angle glaucoma gene (GLC1A).N Engl J Med. 1998; 338: 1022-1027Crossref PubMed Scopus (384) Google Scholar; Morissette et al. Morissette et al., 1998Morissette J Clépet C Moisan S Dubois S Winstall E Vermeeren D Nguyen TD et al.Homozygotes for an autosomal dominant TIGR mutation do not manifest glaucoma.Nat Genet. 1998; 19: 319-321Crossref PubMed Scopus (115) Google Scholar). Because the prevalence of mutations in TIGR/myocilin has not yet been investigated in a large number of pedigrees affected by juvenile- and adult-onset glaucoma, we have performed SSCP and sequence analysis in 152 affected families and in 104 individuals with macular degeneration but with normal intraocular pressures and optic nerves. The pedigrees used for this study are all of North American origin and were ascertained and sampled at the New England Medical Center and the Duke University Medical Center. The diagnostic criteria for POAG included intraocular pressure >22 mm/Hg and glaucomatous optic-nerve damage with consistent visual-field loss. Gonioscopic evaluation showed open angles (at least grade III) without any associated abnormalities. Individuals were identified as affected by adult-onset POAG if onset of the disease occurred after age 35 years, and as affected by juvenile-onset POAG if onset occurred before age 35 years. All pedigrees included in this study had at least two affected individuals. The control population underwent a complete ocular examination and did not show evidence of elevation of intraocular pressure or of optic-nerve disease. For mutation detection, a BAC clone (244L10) containing the TIGR/myocilin gene was identified by the screen of an arrayed BAC library (Research Genetics, Inc.). The BAC DNA was used as a source for the genomic sequence, and three exons separated by two introns were identified. Oligonucleotide primers (sequences available on request from Janey L. Wiggs), developed from the intron sequences flanking the intron/exon boundaries and from the cDNA sequence, were used to selectively amplify overlapping fragments of the coding sequence and the exon/intron splice sites. Sixty-eight families (25 juvenile-onset and 43 adult-onset) were screened for mutations in the entire coding sequence of the gene, and an additional 84 adult-onset families were screened for mutations in the third exon. Abnormal SSCP patterns were observed in 17 of the 152 families screened. To identify sequence variants, direct DNA sequencing was performed bidirectionally, by use of the Amersham Life Science dideoxy sequencing kit. All DNA sequence alterations that resulted in a change of amino acid were found in the third exon of the gene. Deletions, insertions, or splice-site mutations were not found. In the population of individuals without glaucoma, nine individuals had a T→C transition at position 1041, which resulted in a wobble mutation (TYR347TYR). No other base-pair changes were found in the control population. Two different missense mutations were found in 2 of the 25 pedigrees affected by juvenile-onset POAG (table 1). One of these (TYR437HIS) has previously been identified in two pedigrees from North America (Stone et al. Stone et al., 1997Stone EM Fingert JH Alward WLM Nguyen TD Polansky JR Sunden SLF Nishimura D et al.Identification of a gene that causes primary open angle glaucoma.Science. 1997; 275: 668-670Crossref PubMed Scopus (1191) Google Scholar; Alward et al. Alward et al., 1998Alward WLM Fingert JH Coote MA Johnson AT Lerner SF Junqua D Durcan FJ et al.Clinical features associated with mutations in the chromosome 1 open-angle glaucoma gene (GLC1A).N Engl J Med. 1998; 338: 1022-1027Crossref PubMed Scopus (384) Google Scholar). A second mutation (PRO370LEU) was identified in a three-generation POAG pedigree of English ancestry that settled in Maine >100 years ago. This mutation has also been identified in one Japanese family (Suzuki et al. Suzuki et al., 1997Suzuki Y Shirato S Taniguchi F Ohara K Nishimaki K Ohta S Mutations in the TIGR gene in familial primary open-angle glaucoma in Japan.Am J Hum Genet. 1997; 61: 1202-1204Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar) and in two unrelated French families (Adam et al. Adam et al., 1997Adam MF Belmouden A Binisti P Brézin AP Valtot F Béchetoille A Dascotte J-C et al.Recurrent mutations in a single exon encoding the evolutionarily conserved olfactomedin-homology domain of TIGR in familial open-angle glaucoma.Hum Mol Genet. 1997; 6: 2091-2097Crossref PubMed Scopus (247) Google Scholar). The recurrence of this mutation in pedigrees of varied ethnicity suggests that the loss of the proline at this position may severely affect the function of the protein. One juvenile pedigree had a C→T transition at position 366, which resulted in a wobble mutation (122GLY122).Table 1Mutations Identified in Juvenile- and Adult-Onset POAGPOAG Type and PedigreeMutationProband Age at Diagnosis (years)Proband Intraocular Pressure at Diagnosis (mmHg)Juvenile-Onset POAG: 4Pro370Leu (1109 C/T)638 18Tyr437His (1309 T/C)1635Adult-Onset POAG: 27Thr377Met (1131 C/T)4224 125Gln368STOP (1102 C/T)4924 5052Gln368STOP (1102 C/T)7828 5055Gln368STOP (1102 C/T)5331 Open table in a new tab In our analysis, only 8% of juvenile-onset pedigrees had identifiable mutations in the TIGR/myocilin gene. This prevalence is lower than that reported in a study by Adam et al. (Adam et al., 1997Adam MF Belmouden A Binisti P Brézin AP Valtot F Béchetoille A Dascotte J-C et al.Recurrent mutations in a single exon encoding the evolutionarily conserved olfactomedin-homology domain of TIGR in familial open-angle glaucoma.Hum Mol Genet. 1997; 6: 2091-2097Crossref PubMed Scopus (247) Google Scholar) that showed mutations in five of eight pedigrees, demonstrating linkage to the GLC1A locus. In our study, the majority of pedigrees are too small for accurate detection of linkage to any particular locus. The small number of pedigrees, in our study, with mutations in TIGR/myocilin suggests that additional genes are likely to be responsible for this disease. Genetic heterogeneity of juvenile-onset POAG has previously been suggested (Graff et al. Graff et al., 1995Graff C Urbak SF Jerndal T Wadelius C Confirmation of linkage to 1q21-31 in a Danish autosomal dominant juvenile-onset glaucoma family and evidence of genetic heterogeneity.Hum Genet. 1995; 96: 285-289Crossref PubMed Scopus (39) Google Scholar; Wiggs et al. Wiggs et al., 1995Wiggs JL DelBono EA Schuman JS Hutchinson BT Walton DS Clinical features of five pedigrees genetically linked to the juvenile glaucoma locus on chromosome 1q21-q31.Ophthalmology. 1995; 102: 1782-1789Abstract Full Text PDF PubMed Scopus (66) Google Scholar; Avramopoulous et al. Avramopoulos et al., 1996Avramopoulos D Kitsos G Economou-Petersen E Grigoriadou M Vassilopoulos D Papageorgiou C Psilas K et al.Exclusion of one pedigree affected by adult-onset primary open angle glaucoma from linkage to the juvenile glaucoma locus on chromosome 1q221-q31.J Med Genet. 1996; 33: 1043-1044Crossref PubMed Scopus (6) Google Scholar; Richards et al. Richards et al., 1996Richards JE Lichter PR Herman S Hauser ER Hou YC Johnson AT Boehnke M Probable exclusion of GLC1A as a candidate glaucoma gene in a family with middle-age-onset primary open-angle glaucoma.Ophthalmology. 1996; 103: 1035-1040Abstract Full Text PDF PubMed Scopus (14) Google Scholar; WuDunn et al. WuDunn et al., 1996WuDunn D Parrish RK Inana G Genetic heterogeneity in Hispanic families with autosomal dominant juvenile glaucoma.Ophthalmic Genet. 1996; 17: 87-94Crossref PubMed Scopus (1) Google Scholar). Sequence alterations that resulted in a change of amino acid were identified in 5 of 127 families affected by adult-onset POAG. Three of these families had the GLN368STOP mutation, previously reported in pedigrees of North American origin (Stone et al. Stone et al., 1997Stone EM Fingert JH Alward WLM Nguyen TD Polansky JR Sunden SLF Nishimura D et al.Identification of a gene that causes primary open angle glaucoma.Science. 1997; 275: 668-670Crossref PubMed Scopus (1191) Google Scholar; Alward et al. Alward et al., 1998Alward WLM Fingert JH Coote MA Johnson AT Lerner SF Junqua D Durcan FJ et al.Clinical features associated with mutations in the chromosome 1 open-angle glaucoma gene (GLC1A).N Engl J Med. 1998; 338: 1022-1027Crossref PubMed Scopus (384) Google Scholar). The three pedigrees we studied do not have common ancestry. We did not detect the GLN368STOP mutation in any individuals affected by juvenile-onset glaucoma. Two adult-onset pedigrees have sequence alterations that do not segregate with the phenotype. Neither of these sequence changes were seen in 104 control patients. Three members of pedigree 27 had a C→T transition, which resulted in a change of the threonine at amino acid 377 to a methionine. Of the four affected individuals in this family, only three were found to have this alteration. No abnormalities in this gene were found in the remaining affected individual (fig. 1), and it remains a possibility that individual 27-3 is a phenocopy. Pedigree 5039 was found to have a DNA sequence-pair change, which caused the glutamate at position 352 to be replaced by a lysine. Of the four affected individuals in this family, one has the mutation, whereas three do not (fig. 1). Of the four unaffected individuals, one has the mutation but, at age 48 years, does not have any evidence of the disease. This sequence change could represent an extremely rare polymorphism, not present in our study or control populations. In support of this hypothesis, this family is of African American origin, whereas 90% of the study and control populations are Caucasian. The TYR437TYR wobble variant was found in eight adult-onset pedigrees. Previous reports have suggested that 3%–5% of patients with adult-onset glaucoma have mutations in the TIGR/myocilin gene (Stone et al. Stone et al., 1997Stone EM Fingert JH Alward WLM Nguyen TD Polansky JR Sunden SLF Nishimura D et al.Identification of a gene that causes primary open angle glaucoma.Science. 1997; 275: 668-670Crossref PubMed Scopus (1191) Google Scholar; Suzuki et al. Suzuki et al., 1997Suzuki Y Shirato S Taniguchi F Ohara K Nishimaki K Ohta S Mutations in the TIGR gene in familial primary open-angle glaucoma in Japan.Am J Hum Genet. 1997; 61: 1202-1204Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar; Alward et al. Alward et al., 1998Alward WLM Fingert JH Coote MA Johnson AT Lerner SF Junqua D Durcan FJ et al.Clinical features associated with mutations in the chromosome 1 open-angle glaucoma gene (GLC1A).N Engl J Med. 1998; 338: 1022-1027Crossref PubMed Scopus (384) Google Scholar). Our results confirm that mutations in the TIGR/myocilin gene are an uncommon cause of adult-onset POAG. These results are consistent with the current heterogeneity of adult-onset POAG. In recent studies, five loci for adult-onset POAG have been discovered: GLC1B (Stoilova et al. Stoilova et al., 1996Stoilova D Child A Trifan OC Crick RP Coakes RL Sarfarazi M Localization of a locus (GLC1B) for adult-onset primary open angle glaucoma to the 2cen-q13 region.Genomics. 1996; 36: 142-150Crossref PubMed Scopus (197) Google Scholar), GLC1C (Wirtz et al. Wirtz et al., 1997Wirtz MK Samples JR Kramer PL Rust K Topinka JR Yount J Koler RD et al.Mapping a gene for adult-onset primary open-angle glaucoma to chromosome 3q.Am J Hum Genet. 1997; 60: 296-304PubMed Google Scholar), GLC1D (Trifan et al. Trifan et al., 1998Trifan OC Traboulsi EI Stoilova D Alozie I Nguyen R Raja S Sarfarazi M The third locus (GLC1D) for adult-onset primary open-angle glaucoma maps to the 8q23 region.Am J Ophthalmol. 1998; 126: 17-28Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar), GLC1E (Sarfarazi et al. Sarfarazi et al., 1998Sarfarazi M Child A Stoilova D Brice G Desai T Trifan OC Poinoosawmy D et al.Localization of the fourth locus (GLC1E) for adult-onset primary open-angle glaucoma to the 10p15-p14 region.Am J Hum Genet. 1998; 62: 641-652Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar), and GLC1F (Wirtz et al. Wirtz et al., 1998Wirtz MK Samples JR Kramer PL Yount J Rust K Acott TS Identification of a new adult-onset primary open angle glaucoma locus-GLC1F.Invest Ophthalmol Vis Sci. 1998; 39 (abstract 2341): S512Google Scholar). The identification of these and other genes responsible for various forms of glaucoma may lead to valuable insights into the pathophysiology of these important blinding disorders. We thank the families for their willing participation. J.L.W. is supported by NIH grants EY10886 and EY09847, Research to Prevent Blindness, and the Massachusetts Lions; D.V. is suported by NIH grant EY11405, the American Health Assistance Foundation, and the March of Dimes Birth Defects Foundation.

Glaucoma is a progressive blinding disease characterized by gradual loss of vision due to optic neuropathy and retinal ganglion cell death. Increased intraocular pressure is a common feature of glaucoma that is thought to arise from an increased resistance to outflow of aqueous humor through the trabecular meshwork. Mutations of the myocilin gene are one cause of autosomal dominant juvenile- and adult-onset primary open angle glaucoma, but the mechanism by which mutant myocilins cause disease is poorly understood. We have found that disease-causing myocilin mutants are misfolded, are highly aggregation-prone and accumulate in large aggregates in the endoplasmic reticulum (ER) of human embryonic kidney cells and differentiated primary human trabecular meshwork (HTM) cells. In HTM cells, Pro370Leu mutant myocilin is not secreted under normal culture conditions and prolonged expression results in abnormal cell morphology and cell killing. Culturing HTM cells at 30°C, a condition known to facilitate protein folding, promotes secretion of mutant myocilin, normalizes cell morphology and reverses cell lethality. Our results indicate that myocilin-associated glaucoma is an ER storage disease and suggest a progression of events in which chronic expression of misfolded, non-secreted myocilin leads to HTM cell death, trabecular meshwork dysfunction and, ultimately, a dominant glaucoma phenotype. The beneficial effects of facilitating folding and secretion of mutant myocilin suggest a new type of treatment for this form of glaucoma.

Some insight into human evolution has been gained from the sequencing of four Y chromosome genes. Primary genomic sequencing determined gene SMCY to be composed of 27 exons that comprise 4,620 bp of coding sequence. The unfinished sequencing of the 5′ portion of gene UTY1 was completed by primer walking, and a total of 20 exons were found. By using denaturing HPLC, these two genes, as well as DBY and DFFRY , were screened for polymorphic sites in 53–72 representatives of the five continents. A total of 98 variants were found, yielding nucleotide diversity estimates of 2.45 × 10 −5 , 5.07 × 10 −5 , and 8.54 × 10 −5 for the coding regions of SMCY , DFFRY , and UTY1 , respectively, with no variant having been observed in DBY . In agreement with most autosomal genes, diversity estimates for the noncoding regions were about 2- to 3-fold higher and ranged from 9.16 × 10 −5 to 14.2 × 10 −5 for the four genes. Analysis of the frequencies of derived alleles for all four genes showed that they more closely fit the expectation of a Luria–Delbrück distribution than a distribution expected under a constant population size model, providing evidence for exponential population growth. Pairwise nucleotide mismatch distributions date the occurrence of population expansion to ≈28,000 years ago. This estimate is in accord with the spread of Aurignacian technology and the disappearance of the Neanderthals.

Unequal crossing-over within a head-to-tail tandem array of the homologous red and green visual pigment genes has been proposed to explain the observed variation in green-pigment gene number among individuals and the prevalence of red-green fusion genes among color-blind subjects. This model was tested by probing the structure of the red and green pigment loci with long-range physical mapping techniques. The loci were found to constitute a gene array with an approximately 39-kilobase repeat length. The position of the red pigment gene at the 5' edge of the array explains its lack of variation in copy number. Restriction maps of the array in four individuals who differ in gene number are consistent with a head-to-tail configuration of the genes. These results provide physical evidence in support of the model and help to explain the high incidence of color blindness in the human population.

To screen a population with primary open-angle glaucoma for mutations in the gene that encodes the trabecular meshwork inducible glucocorticoid response protein (TIGR), also known as myocilin (MYOC).Ophthalmologic information was collected for study subjects with primary open-angle glaucoma and their relatives. Mutation screening of 74 primary open-angle glaucoma probands was conducted by sequencing TIGR/MYOC coding sequence and splice sites.In 23 families we detected 13 nonsynonymous sequence changes, nine of which appear to be mutations likely to cause or contribute to primary open-angle glaucoma. Two mutations, Arg272Gly and Ile499Ser, and one nonsynonymous sequence variant, Asn57Asp, are novel. We found mutations in nine of 25 juvenile glaucoma probands (36%) and two of 49 adult-onset glaucoma probands (4%). Age classification of families rather than individual probands revealed mutations in three of nine families with strictly juvenile primary open-angle glaucoma (33%), and no mutations in 39 families with strictly adult-onset primary open-angle glaucoma (0%). In families with mixed-onset primary open-angle glaucoma containing both juvenile primary open-angle glaucoma and adult-onset primary open-angle glaucoma cases, we found mutations in eight of 26 families (31%).Our data suggest that Gly252Arg, Arg272Gly, Glu323Lys, Gln368STOP, Pro370Leu, Thr377Met, Val426Phe, Ile477Asn, and Ile499Ser are likely to play roles that cause or contribute to the etiology of autosomal dominant primary open-angle glaucoma. Our finding of more TIGR/MYOC mutations in families with mixed-onset primary open-angle glaucoma than in the families with strictly adult-onset primary open-angle glaucoma implies that the presence of relatives with juvenile primary open-angle glaucoma in a family could be used as a basis for identifying a subset of the population with adult-onset primary open-angle glaucoma with higher prevalence of TIGR/MYOC mutations. To address this issue, and to refine estimations of mutation prevalence in these age-defined subpopulations, prospective study of a larger population ascertained entirely through adult-onset primary open-angle glaucoma probands will be needed.

Posterior polymorphous corneal dystrophy (PPCD, also known as PPMD) is a rare disease involving metaplasia and overgrowth of corneal endothelial cells. In patients with PPCD, these cells manifest in an epithelial morphology and gene expression pattern, produce an aberrant basement membrane, and, sometimes, spread over the iris and nearby structures in a way that increases the risk for glaucoma. We previously mapped PPCD to a region (PPCD3) on chromosome 10 containing the gene that encodes the two-handed zinc-finger homeodomain transcription factor TCF8. Here, we report a heterozygous frameshift mutation in TCF8 that segregates with PPCD in the family used to map PPCD3 and four different heterozygous nonsense and frameshift mutations in TCF8 in four other PPCD probands. Family reports of inguinal hernia, hydrocele, and possible bone anomalies in affected individuals suggest that individuals with TCF8 mutations should be examined for nonocular anomalies. We detect transcripts of all three identified PPCD genes (VSX1, COL8A2, and TCF8) in the cornea. We show presence of a complex (core plus secondary) binding site for TCF8 in the promoter of Alport syndrome gene COL4A3, which encodes collagen type IV α3, and we present immunohistochemical evidence of ectopic expression of COL4A3 in corneal endothelium of the proband of the original PPCD3 family. Identification of TCF8 as the PPCD3 gene provides a valuable tool for the study of critical gene regulation events in PPCD pathology and suggests a possible role for TCF8 mutations in altered structure and function of cells lining body cavities other than the anterior chamber of the eye. Thus, this study has identified TCF8 as the gene responsible for approximately half of the cases of PPCD, has implicated TCF8 mutations in developmental abnormalities outside the eye, and has presented the TCF8 regulatory target, COL4A3, as a key, shared molecular component of two different diseases, PPCD and Alport syndrome. Posterior polymorphous corneal dystrophy (PPCD, also known as PPMD) is a rare disease involving metaplasia and overgrowth of corneal endothelial cells. In patients with PPCD, these cells manifest in an epithelial morphology and gene expression pattern, produce an aberrant basement membrane, and, sometimes, spread over the iris and nearby structures in a way that increases the risk for glaucoma. We previously mapped PPCD to a region (PPCD3) on chromosome 10 containing the gene that encodes the two-handed zinc-finger homeodomain transcription factor TCF8. Here, we report a heterozygous frameshift mutation in TCF8 that segregates with PPCD in the family used to map PPCD3 and four different heterozygous nonsense and frameshift mutations in TCF8 in four other PPCD probands. Family reports of inguinal hernia, hydrocele, and possible bone anomalies in affected individuals suggest that individuals with TCF8 mutations should be examined for nonocular anomalies. We detect transcripts of all three identified PPCD genes (VSX1, COL8A2, and TCF8) in the cornea. We show presence of a complex (core plus secondary) binding site for TCF8 in the promoter of Alport syndrome gene COL4A3, which encodes collagen type IV α3, and we present immunohistochemical evidence of ectopic expression of COL4A3 in corneal endothelium of the proband of the original PPCD3 family. Identification of TCF8 as the PPCD3 gene provides a valuable tool for the study of critical gene regulation events in PPCD pathology and suggests a possible role for TCF8 mutations in altered structure and function of cells lining body cavities other than the anterior chamber of the eye. Thus, this study has identified TCF8 as the gene responsible for approximately half of the cases of PPCD, has implicated TCF8 mutations in developmental abnormalities outside the eye, and has presented the TCF8 regulatory target, COL4A3, as a key, shared molecular component of two different diseases, PPCD and Alport syndrome.

A technique is described for physically positioning any cloned DNA on a native or artificial Saccharomyces cerevisiae chromosome. The technique involves splitting a chromosome at a specific site by transformation with short linear molecules containing the cloned DNA at one end and telomeric sequences at the other. Recombination between the end of the linear molecules and homologous chromosomal sequences gives rise to chromosome fragments comprising all sequences distal or proximal to the mapping site depending on the orientation of the cloned DNA. The recombinant products are recovered by screening for stabilization of a suppressor tRNA on the linear molecules using a colony color assay. The cloned DNA is positioned relative to the chromosome ends by sizing the chromosomal fragments using alternating contour-clamped homogeneous electric field gel electrophoresis. Application of this technique to organisms other than S. cerevisiae and to the analysis of exogenous DNA cloned in yeast is discussed.

Retinitis pigmentosa (RP), a disease characterized by progressive loss of photoreceptors, exhibits significant genetic heterogeneity. Several genes associated with U4/U6–U5 triple small nuclear ribonucleoprotein (tri-snRNP) complex of the spliceosome have been implicated in autosomal dominant RP (adRP). HPrp4, encoded by PRPF4, regulates the stability of U4/U6 di-snRNP, which is essential for continuous splicing. Here, we identified two heterozygous variants in PRPF4, including c.-114_-97del in a simplex RP patient and c.C944T (p.Pro315Leu), which co-segregates with disease phenotype in a family with adRP. Both variants were absent in 400 unrelated controls. The c.-114_-97del, predicted to affect two transcription factor binding sites, was shown to down-regulate the promoter activity of PRPF4 by a luciferase assay, and was associated with a significant reduction of PRPF4 expression in the blood cells of the patient. In fibroblasts from an affected individual with the p.Pro315Leu variant, the expression levels of several tri-snRNP components, including PRPF4 itself, were up-regulated, with altered expression pattern of SC35, a spliceosome marker. The same alterations were also observed in cells over expressing hPrp4Pro315Leu, suggesting that they arose as a compensatory response to a compromised splicing mechanism caused by hPrp4 dysfunction. Further, over expression of hPrp4Pro315Leu, but not hPrp4WT, triggered systemic deformities in wild-type zebrafish embryos with the retina primarily affected, and dramatically augmented death rates in morphant embryos, in which orthologous zebrafish prpf4 gene was silenced. We conclude that mutations of PRPF4 cause RP via haploinsufficiency and dominant-negative effects, and establish PRPF4 as a new U4/U6–U5 snRNP component associated with adRP.

The CAV1/CAV2 (caveolin 1 and caveolin 2) genomic region previously was associated with primary open-angle glaucoma (POAG), although replication among independent studies has been variable. The aim of this study was to assess the association between CAV1/CAV2 single nucleotide polymorphisms (SNPs) and POAG in a large case-control dataset and to explore associations by gender and pattern of visual field (VF) loss further.Case-control study.We analyzed 2 large POAG data sets: the Glaucoma Genes and Environment (GLAUGEN) study (976 cases, 1140 controls) and the National Eye Institute Glaucoma Human Genetics Collaboration (NEIGHBOR) consortium (2132 cases, 2290 controls).We studied the association between 70 SNPs located within the CAV1/CAV2 genomic region in the GLAUGEN and NEIGHBOR studies, both genotyped on the Illumina Human 660WQuadv1C BeadChip array and imputed with the Markov Chain Haplotyping algorithm using the HapMap 3 reference panel. We used logistic regression models of POAG in the overall population and separated by gender, as well as by POAG subtypes defined by type of VF defect (peripheral or paracentral). Results from GLAUGEN and NEIGHBOR were meta-analyzed, and a Bonferroni-corrected significance level of 7.7 × 10(-4) was used to account for multiple comparisons.Overall POAG, overall POAG by gender, and POAG subtypes defined by pattern of early VF loss.We found significant associations between 10 CAV1/CAV2 SNPs and POAG (top SNP, rs4236601; pooled P = 2.61 × 10(-7)). Of these, 9 were significant only in women (top SNP, rs4236601; pooled P = 1.59 × 10(-5)). Five of the 10 CAV1/CAV2 SNPs were associated with POAG with early paracentral VF (top SNP, rs17588172; pooled P = 1.07 × 10(-4)), and none of the 10 were associated with POAG with peripheral VF loss only or POAG among men.CAV1/CAV2 SNPs were associated significantly with POAG overall, particularly among women. Furthermore, we found an association between CAV1/CAV2 SNPs and POAG with paracentral VF defects. These data support a role for caveolin 1, caveolin 2, or both in POAG and suggest that the caveolins particularly may affect POAG pathogenesis in women and in patients with early paracentral VF defects.

Glaucoma is characterized by irreversible optic nerve degeneration and is the most frequent cause of irreversible blindness worldwide. Here, the International Glaucoma Genetics Consortium conducts a meta-analysis of genome-wide association studies of vertical cup-disc ratio (VCDR), an important disease-related optic nerve parameter. In 21,094 individuals of European ancestry and 6,784 individuals of Asian ancestry, we identify 10 new loci associated with variation in VCDR. In a separate risk-score analysis of five case-control studies, Caucasians in the highest quintile have a 2.5-fold increased risk of primary open-angle glaucoma as compared with those in the lowest quintile. This study has more than doubled the known loci associated with optic disc cupping and will allow greater understanding of mechanisms involved in this common blinding condition.

Glaucoma is a leading cause of blindness worldwide. Primary open-angle glaucoma (POAG) is the most common subtype and is a complex trait with multigenic inheritance. Genome-wide association studies have previously identified a significant association between POAG and the SIX6 locus (rs10483727, odds ratio (OR) = 1.32, p = 3.87×10−11). SIX6 plays a role in ocular development and has been associated with the morphology of the optic nerve. We sequenced the SIX6 coding and regulatory regions in 262 POAG cases and 256 controls and identified six nonsynonymous coding variants, including five rare and one common variant, Asn141His (rs33912345), which was associated significantly with POAG (OR = 1.27, p = 4.2×10−10) in the NEIGHBOR/GLAUGEN datasets. These variants were tested in an in vivo Danio rerio (zebrafish) complementation assay to evaluate ocular metrics such as eye size and optic nerve structure. Five variants, found primarily in POAG cases, were hypomorphic or null, while the sixth variant, found only in controls, was benign. One variant in the SIX6 enhancer increased expression of SIX6 and disrupted its regulation. Finally, to our knowledge for the first time, we have identified a clinical feature in POAG patients that appears to be dependent upon SIX6 genotype: patients who are homozygous for the SIX6 risk allele (His141) have a statistically thinner retinal nerve fiber layer than patients homozygous for the SIX6 non-risk allele (Asn141). Our results, in combination with previous SIX6 work, lead us to hypothesize that SIX6 risk variants disrupt the development of the neural retina, leading to a reduced number of retinal ganglion cells, thereby increasing the risk of glaucoma-associated vision loss.

Central corneal thickness (CCT) is a highly heritable trait associated with complex eye diseases such as keratoconus and glaucoma. We perform a genome-wide association meta-analysis of CCT and identify 19 novel regions. In addition to adding support for known connective tissue-related pathways, pathway analyses uncover previously unreported gene sets. Remarkably, >20% of the CCT-loci are near or within Mendelian disorder genes. These included FBN1, ADAMTS2 and TGFB2 which associate with connective tissue disorders (Marfan, Ehlers-Danlos and Loeys-Dietz syndromes), and the LUM-DCN-KERA gene complex involved in myopia, corneal dystrophies and cornea plana. Using index CCT-increasing variants, we find a significant inverse correlation in effect sizes between CCT and keratoconus (r = -0.62, P = 5.30 × 10-5) but not between CCT and primary open-angle glaucoma (r = -0.17, P = 0.2). Our findings provide evidence for shared genetic influences between CCT and keratoconus, and implicate candidate genes acting in collagen and extracellular matrix regulation.

Uniparental disomy (UPD) is a rare condition in which a diploid offspring carries a chromosomal pair from a single parent. We now report the first two cases of UPD resulting in retinal degeneration. We identified an apparently homozygous loss-of-function mutation of RPE65 (1p31) in one retinal dystrophy patient and an apparently homozygous loss-of-function mutation of MERTK (2q14.1) in a second retinal dystrophy patient. In both families, the gene defect was present in the patient's heterozygous father but not in the patient's mother. Analysis of haplotypes in each nuclear kindred, by use of DNA polymorphisms distributed along both chromosomal arms, indicated the absence of the maternal allele for all informative markers tested on chromosome 1 in the first patient and on chromosome 2 in the second patient. Our results suggest that retinal degeneration in these individuals is due to apparently complete paternal isodisomy involving reduction to homoallelism for RPE65 or MERTK loss-of-function alleles. Our findings provide evidence for the first time, in the case of chromosome 2, and confirm previous observations, in the case of chromosome 1, that there are no paternally imprinted genes on chromosomes 1 and 2 that have a major effect on phenotype.

We report the discovery of a polymorphic A to G transition found on the human Y chromosome by sequencing Y-specific sequence-tagged sites (STSs). It shows maximal linkage disequilibrium with a previously described Alu insertional polymorphism. We analyze further an apparently African Y chromosome which seems to have entered a Mexican Mayan population several generations ago. Using the newly discovered transition and the Y-speclfic polymorphic Alu insertion, we discuss how the chromosome's haplotype information might be used to answer questions of houman origins and migrations.

Glaucoma is a blinding eye disease that affects ∼70 000 000 people world-wide. Mutations in the gene TIGR/MYOC have been shown to cause the most common form of the disease, primary open angle glaucoma, in selected families. Amino acid sequence variants of the gene have been found in 2–4% of sporadic primary open angle glaucoma cases. Most variants are rare and it is often difficult to definitively distinguish between a deleterious mutation and a benign variant solely on the basis of relative frequencies in patient and control groups. The function of the TIGR/myocilin protein is unknown and an assay to functionally classify variants is lacking. We sought to develop a biochemical assay to distinguish different forms of TIGR/myocilin. We investigated the Triton X-100 detergent solubility characteristics of mutant and normal forms of the protein, expressed by transfection in cultured cells. We observed a clear difference in the behavior of the two types of TIGR/myocilin; all confirmed mutant proteins tested were substantially Triton insoluble, while normal protein and controls were completely soluble. We also tested seven ambiguous variant proteins and classified them as mutant or normal on the basis of their Triton solubility. The results in some cases validated, and in other cases contradicted, earlier classifications of these variants. To our knowledge, Triton solubility is the first example of a general difference in the properties of mutant and normal forms of TIGR/myocilin. The assay we have developed will be useful for discerning protein functional information from the location of mutations, will aid genetic counseling of individuals with TIGR/myocilin variants and may provide a clue to understanding a mechanism by which mutations in TIGR/MYOC cause glaucoma.

<h3>Summary</h3> Nanophthalmos is an uncommon developmental ocular disorder characterized by a small eye, as indicated by short axial length, high hyperopia (severe farsightedness), high lens/eye volume ratio, and a high incidence of angle-closure glaucoma. We performed clinical and genetic evaluations of members of a large family in which nanophthalmos is transmitted in an autosomal dominant manner. Ocular examinations of 22 affected family members revealed high hyperopia (range +7.25–+13.00 diopters; mean +9.88 diopters) and short axial length (range 17.55–19.28 mm; mean 18.13 mm). Twelve affected family members had angle-closure glaucoma or occludable anterior-chamber angles. Linkage analysis of a genome scan demonstrated highly significant evidence that nanophthalmos in this family is the result of a defect in a previously unidentified locus (<i>NNO1</i>) on chromosome 11. The gene was localized to a 14.7-cM interval between D11S905 and D11S987, with a maximum LOD score of 5.92 at a recombination fraction of .00 for marker D11S903 and a multipoint maximum LOD score of 6.31 for marker D11S1313. <i>NNO1</i> is the first human locus associated with nanophthalmos or with an angle-closure glaucoma phenotype, and the identification of the <i>NNO1</i> locus is the first step toward the cloning of the gene. A cloned copy of the gene will enable examination of the relationship, if any, between nanophthalmos and less severe forms of hyperopia and between nanophthalmos and other conditions in which angle-closure glaucoma is a feature.

Purpose To assess the association between single nucleotide polymorphisms (SNPs) of the gene region containing cyclin-dependent kinase inhibitor 2B antisense noncoding RNA (CDKN2B-AS1) and glaucoma features among primary open-angle glaucoma (POAG) patients. Design Retrospective observational case series. Methods We studied associations between 10 CDKN2B-AS1 SNPs and glaucoma features among 976 POAG cases from the Glaucoma Genes and Environment (GLAUGEN) study and 1971 cases from the National Eye Institute Glaucoma Human Genetics Collaboration (NEIGHBOR) consortium. For each patient, we chose the feature from the eye with the higher value. We created cohort-specific multivariable models for glaucoma features and then meta-analyzed the results. Results For 9 of the 10 protective CDKN2B-AS1 SNPs with minor alleles associated with reduced disease risk (eg, the G allele at rs2157719), POAG patients carrying these minor alleles had smaller cup-to-disc ratio (0.05 units smaller per G allele at diagnosis; 95% CI: −0.08, −0.03; P = 6.23E-05) despite having higher intraocular pressure (IOP) (0.70 mm Hg higher per G allele at DNA collection; 95% CI: 0.40, 1.00; P = 5.45E-06). For the 1 adverse rs3217992 SNP with minor allele A associated with increased disease risk, POAG patients with A alleles had larger cup-to-disc ratio (0.05 units larger per A allele at diagnosis; 95% CI: 0.02, 0.07; P = 4.74E-04) despite having lower IOP (−0.57 mm Hg per A allele at DNA collection; 95% CI: −0.84, −0.29; P = 6.55E-05). Conclusion Alleles of CDKN2B-AS1 SNPs, which influence risk of developing POAG, also modulate optic nerve degeneration among POAG patients, underscoring the role of CDKN2B-AS1 in POAG. To assess the association between single nucleotide polymorphisms (SNPs) of the gene region containing cyclin-dependent kinase inhibitor 2B antisense noncoding RNA (CDKN2B-AS1) and glaucoma features among primary open-angle glaucoma (POAG) patients. Retrospective observational case series. We studied associations between 10 CDKN2B-AS1 SNPs and glaucoma features among 976 POAG cases from the Glaucoma Genes and Environment (GLAUGEN) study and 1971 cases from the National Eye Institute Glaucoma Human Genetics Collaboration (NEIGHBOR) consortium. For each patient, we chose the feature from the eye with the higher value. We created cohort-specific multivariable models for glaucoma features and then meta-analyzed the results. For 9 of the 10 protective CDKN2B-AS1 SNPs with minor alleles associated with reduced disease risk (eg, the G allele at rs2157719), POAG patients carrying these minor alleles had smaller cup-to-disc ratio (0.05 units smaller per G allele at diagnosis; 95% CI: −0.08, −0.03; P = 6.23E-05) despite having higher intraocular pressure (IOP) (0.70 mm Hg higher per G allele at DNA collection; 95% CI: 0.40, 1.00; P = 5.45E-06). For the 1 adverse rs3217992 SNP with minor allele A associated with increased disease risk, POAG patients with A alleles had larger cup-to-disc ratio (0.05 units larger per A allele at diagnosis; 95% CI: 0.02, 0.07; P = 4.74E-04) despite having lower IOP (−0.57 mm Hg per A allele at DNA collection; 95% CI: −0.84, −0.29; P = 6.55E-05). Alleles of CDKN2B-AS1 SNPs, which influence risk of developing POAG, also modulate optic nerve degeneration among POAG patients, underscoring the role of CDKN2B-AS1 in POAG.

Purpose.: To generate and characterize a constitutively active, RPE-specific, cre-expressing transgenic mouse line. This line can be used to create RPE-specific knockouts by crossing with mice harboring loxP-flanked (floxed) genes. Methods.: A transgene construct was assembled with the BEST1 promoter driving cre expression. Transgenic mice were generated on a C57BL/6 background. Cre expression was assessed by immunofluorescence and Western blot analysis. Cre enzymatic activity was tested by crossing to three lines with floxed DNA regions and detecting deletion of the intervening sequences or through histochemical detection of lacZ activity. Potential cre-mediated toxicity was assessed by retinal histology up to 24 months of age and by electroretinography. Results.: The BEST1-cre line with expression in the highest percentage of RPE cells displayed a patchy mosaic expression pattern, with 50% to 90% of RPE cells expressing cre. In mice outcrossed to a mixed B6/129 background, expression was consistently found in 90% of RPE cells. Within the eye, only the RPE cells were immunoreactive with an anti-cre antibody. Maximum cre expression quantified by Western blot analysis occurred at P28. Crosses with three lines containing floxed sequences revealed RPE-specific cre activity in the eye and extraocular expression limited to the testes. Histology and electroretinography showed no cre-mediated RPE toxicity. Conclusions.: This BEST1-cre transgenic line enables generation of RPE-specific knockout mice. The mosaic expression pattern provides an internal control; the non–cre-expressing RPE cells continue to express the floxed genes. These mice should facilitate study of the multifunctional RPE and the generation of mouse models of human retinal disease.

Purpose.: Hereditary retinal dystrophies (HRDs) are a group of monogenic diseases characterized by an irreversible loss of photoreceptors. HRDs exhibit significant genetic and clinical heterogeneities challenging traditional techniques for determining disease-causal mutations. This study aims to develop an efficient molecular diagnostic platform for HRDs, and to determine the genetic basis for 25 randomly collected Chinese families with a variety of HRDs. Methods.: We designed a high throughput sequence capture microarray targeting 179 genes associated with HRDs and 10 candidate genes. We combined sequence capture with next-generation sequencing (NGS) to screen for mutations in the cohort of Chinese families. Variants detected by NGS were filtered, validated, and prioritized by pathogenicity analysis. Genotypes and phenotypes were correlated. Results.: We identified four recurrent single mutations, two compound mutations, and eight novel putative causative mutations, including five putative pathogenic alleles (e.g., premature stop codons and frame shifts) and three novel missense variants that are very likely pathogenic. These findings provided specific genetic diagnoses in 14 of 25 families (56%). Among these, identification of a mutation in VCAN in a family with a complicated phenotype helped to finalize the clinical diagnosis as Wagner syndrome. In another five families, 11 potential novel pathogenic variants were identified. Conclusions.: A substantial number of potential new genes and new mutations associated with HRDs remain to be discovered. Identification of the novel HRDs-causing mutations in our study not only provides a better understanding of genotype–phenotype relationships in these diseases, but also demonstrates that the approach described herein is an effective method for large scale mutation detection among diverse and complicated HRDs cases.

Derangements of the blood-brain barrier (BBB) or blood-retinal barrier (BRB) occur in disorders ranging from stroke, cancer, diabetic retinopathy, and Alzheimer's disease. The Norrin/FZD4/TSPAN12 pathway activates WNT/β-catenin signaling, which is essential for BBB and BRB function. However, systemic pharmacologic FZD4 stimulation is hindered by obligate palmitoylation and insolubility of native WNTs and suboptimal properties of the FZD4-selective ligand Norrin. Here, we develop L6-F4-2, a non-lipidated, FZD4-specific surrogate which significantly improves subpicomolar affinity versus native Norrin. In Norrin knockout (NdpKO) mice, L6-F4-2 not only potently reverses neonatal retinal angiogenesis deficits, but also restores BRB and BBB function. In adult C57Bl/6J mice, post-stroke systemic delivery of L6-F4-2 strongly reduces BBB permeability, infarction, and edema, while improving neurologic score and capillary pericyte coverage. Our findings reveal systemic efficacy of a bioengineered FZD4-selective WNT surrogate during ischemic BBB dysfunction, with potential applicability to adult CNS disorders characterized by an aberrant blood-brain barrier.

To determine if a patient with an interstitial deletion of chromosome 1 is hemizygous for the TIGR/MYOC gene and if that patient has glaucoma.A patient with an interstitial deletion of chromosome 1 was clinically examined for evidence of glaucoma. DNA samples from the patient and her family were used for molecular studies to determine the boundaries of the chromosome 1 deletion using polymorphic markers located on chromosome 1q21 to 1q24. Additional markers located in the vicinity of the TIGR/MYOC gene, including 2 derived from the ends of the gene, were used to determine if it was included in the deletion.The patient and her family showed no evidence of glaucoma. Molecular analysis demonstrated that a complex deletion of the maternal copy of chromosome 1 included the entire TIGR/MYOC gene.We have determined that the patient has only 1 functional copy of TIGR/MYOC. The lack of clinical evidence of glaucoma suggests that haploinsufficiency of the TIGR/MYOC protein is not the cause of early-onset glaucoma associated with mutations in TIGR/MYOC.Missense and nonsense mutations in the TIGR/MYOC gene have been associated with juvenile- and adult-onset primary open-angle glaucoma. Although many different mutations have been correlated with the disease, the underlying genetic mechanism (haploinsufficiency, gain of function, or a dominant negative effect) remains unknown. Information regarding the genetic mechanism responsible for TIGR/MYOC-associated glaucoma is necessary for further studies designed to develop transgenic animal models and gene-related therapy.

purpose. Gene therapy has shown promise in animal models of retinal disease, with the most success achieved to date with viral vectors used for gene delivery. Viral vectors, however, have side effects and limitations and are difficult to manufacture. The present study was conducted in an attempt to develop a novel system for long-term gene transfer in rat retinal pigment epithelium (RPE), by using nonviral transfection methods for gene transfer and the integrase from the bacteriophage φC31 to confer long-term gene expression by means of genomic integration. methods. Efficient nonviral delivery of plasmid DNA to rat RPE in vivo was achieved by using subretinal injection of plasmid DNA, followed by in situ electroporation. Gene delivery was evaluated by analyzing enhanced green fluorescent protein (eGFP) expression in frozen sections. In subsequent experiments, a plasmid expressing luciferase, with or without a plasmid encoding the φC31 integrase, was delivered to rat RPE. Luciferase expression was followed over time by using in vivo luciferase imaging. results. Subretinal injection followed by electroporation yielded abundant transgene expression in the rat RPE. Expression was strongest 48 hours after delivery. In the absence of φC31 integrase, transgene expression declined to near-background levels within 3 to 4 weeks after treatment. By contrast, coinjection of the integrase plasmid led to long-term stable transgene expression throughout the 4.5-month test period. Eyes injected with φC31 integrase showed ∼85-fold higher long-term transgene expression in the retina than eyes without integrase. conclusions. Subretinal injection of DNA followed by electroporation affords abundant transfer of plasmid DNA in rat RPE. φC31 integrase confers robust long-term transgene expression by mediating genomic integration of the transgene. These findings suggest that φC31 integrase may be a simple and effective tool for nonviral long-term gene transfer in the eye.

Topical use of dexamethasone has long been associated with steroid induced-glaucoma, although the mechanism is unknown. We applied a strict filtering of comparative microarray data to more than 18,000 genes to evaluate global gene expression of cultured human trabecular meshwork cells in response to treatment with dexamethasone.Three human trabecular meshwork cell primary cultures from nonglaucomatous donors were incubated with and without dexamethasone for 21 days. Relative gene expression was evaluated by analysis of U133A GeneChip and the results validated using quantitative polymerase chain reaction (PCR).Application of strict filtering to include only genes with statistically significant differences in gene expression across all three trabecular meshwork cell cultures produced a list of 1,260 genes. Significant changes in signal level were observed, including 23 upregulated and 18 downregulated genes that changed greater than three fold in each of three cell cultures. Using quantitative PCR we found changes greater than a thousand fold for two genes (SLP1 and SAA2) and changes greater than a hundred fold for another five genes (ANGPTL7, MYOC, SAA1, SERPINA3, and ZBTB16).Expression changes in trabecular meshwork cells in response to dexamethasone treatment indicate that a group of actins and actin-associated proteins are involved in the development of cross-linked actin networks that form in response to dexamethasone. A trend was identified toward decreased expression of protease genes accompanied by an increased expression of protease inhibitors. Such a trend in nonproteasomal proteolysis conceivably affects gene product levels above the level of transcription. Only two genes, MYOC and IGFBP2, showed significantly elevated expression after dexamethasone treatment in our study and the other three previously published reports of primary culture trabecular meshwork cell gene expression.

Mertk is a key phagocytic receptor in the immune, male reproductive, and visual systems. In the retinal pigment epithelium, Mertk is required for the daily ingestion of photoreceptor outer segment (OS) tips. Loss of Mertk function causes retinal degeneration in rats, mice, and humans; however, little is known about the mechanism by which Mertk regulates the ingestion phase of retinal pigment epithelial (RPE) phagocytosis. To address this, the authors sought proteins that associated with Mertk during OS phagocytosis.Lysates of RPE-J cells challenged with OS for various times were immunoprecipitated with Mertk antibody. Potential interacting proteins were identified by mass spectrometry and characterized with confocal microscopy, pharmacologic inhibition, and siRNA knockdown coupled with an in vitro phagocytic assay in primary RPE cells.Myh9, the non-muscle myosin II-A heavy chain, was enriched in immunoprecipitates from OS-treated samples. Myosin II-A and II-B isoforms exhibited a striking redistribution in wild-type rat primary RPE cells challenged with OS, moving from the cell periphery to colocalize with ingested OS over time. In contrast, myosin II-A redistribution in response to OS was blunted in primary RPE cells from RCS rats, which lack functional Mertk. Wild-type rat primary RPE cells treated with the myosin II-specific inhibitor blebbistatin or myosin II siRNAs exhibited a significant phagocytic defect.Mertk mobilizes myosin II from the RPE cell periphery to sites of OS engulfment, where myosin II function is essential for the normal phagocytic ingestion of OS.

Primary open-angle glaucoma (POAG) is a common disease with complex inheritance. The identification of genes predisposing to POAG is an important step toward the development of novel gene-based methods of diagnosis and treatment. Genome-wide association studies (GWAS) have successfully identified genes contributing to complex traits such as POAG however, such studies frequently require very large sample sizes, and thus, collaborations and consortia have been of critical importance for the GWAS approach. In this report we describe the formation of the NEIGHBOR consortium, the harmonized case control definitions used for a POAG GWAS, the clinical features of the cases and controls, and the rationale for the GWAS study design.

To investigate the effects of central corneal thickness (CCT)-associated variants on primary open-angle glaucoma (POAG) risk using single nucleotide polymorphisms (SNP) data from the Glaucoma Genes and Environment (GLAUGEN) and National Eye Institute (NEI) Glaucoma Human Genetics Collaboration (NEIGHBOR) consortia.A replication analysis of previously reported CCT SNPs was performed in a CCT dataset (n = 1117) and these SNPs were then tested for association with POAG using a larger POAG dataset (n = 6470). Then a CCT genome-wide association study (GWAS) was performed. Top SNPs from this analysis were selected and tested for association with POAG. cDNA libraries from fetal and adult brain and ocular tissue samples were generated and used for candidate gene expression analysis.Association with one of 20 previously published CCT SNPs was replicated: rs12447690, near the ZNF469 gene (P = 0.001; β = -5.08 μm/allele). None of these SNPs were significantly associated with POAG. In the CCT GWAS, no SNPs reached genome-wide significance. After testing 50 candidate SNPs for association with POAG, one SNP was identified, rs7481514 within the neurotrimin (NTM) gene, that was significantly associated with POAG in a low-tension subset (P = 0.00099; Odds Ratio [OR] = 1.28). Additionally, SNPs in the CNTNAP4 gene showed suggestive association with POAG (top SNP = rs1428758; P = 0.018; OR = 0.84). NTM and CNTNAP4 were shown to be expressed in ocular tissues.The results suggest previously reported CCT loci are not significantly associated with POAG susceptibility. By performing a quantitative analysis of CCT and a subsequent analysis of POAG, SNPs in two cell adhesion molecules, NTM and CNTNAP4, were identified and may increase POAG susceptibility in a subset of cases.

Inherited photoreceptor degenerations (IPDs) are the most genetically heterogeneous of Mendelian diseases. Many IPDs exhibit substantial phenotypic variability, but the basis is usually unknown. Mutations in MERTK cause recessive IPD phenotypes associated with the RP38 locus. We have identified a murine genetic modifier of Mertk-associated photoreceptor degeneration, the C57BL/6 (B6) allele of which acts as a suppressor. Photoreceptors degenerate rapidly in Mertk-deficient animals homozygous for the 129P2/Ola (129) modifier allele, whereas animals heterozygous for B6 and 129 modifier alleles exhibit an unusual intermixing of degenerating and preserved retinal regions, with females more severely affected than males. Mertk-deficient mice homozygous for the B6 modifier allele display degeneration only in the far periphery, even at 8 months of age, and have improved retinal function compared to animals homozygous for the 129 allele. We genetically mapped the modifier to an approximately 2-megabase critical interval that includes Tyro3, a paralog of Mertk. Tyro3 expression in the outer retina varies with modifier genotype in a manner characteristic of a cis-acting expression quantitative trait locus (eQTL), with the B6 allele conferring an approximately three-fold higher expression level. Loss of Tyro3 function accelerates the pace of photoreceptor degeneration in Mertk knockout mice, and TYRO3 protein is more abundant in the retinal pigment epithelium (RPE) adjacent to preserved central retinal regions of Mertk knockout mice homozygous for the B6 modifier allele. Endogenous human TYRO3 protein co-localizes with nascent photoreceptor outer segment (POS) phagosomes in a primary RPE cell culture assay, and expression of murine Tyro3 in cultured cells stimulates phagocytic ingestion of POS. Our findings demonstrate that Tyro3 gene dosage modulates Mertk-associated retinal degeneration, provide strong evidence for a direct role for TYRO3 in RPE phagocytosis, and suggest that an eQTL can modify a recessive IPD.

Myocilin is a protein found in the extracellular matrix of trabecular meshwork tissue, the anatomical region of the eye involved in regulating intraocular pressure. Wild-type (WT) myocilin has been associated with steroid-induced glaucoma, and variants of myocilin have been linked to early-onset inherited glaucoma. Elevated levels and aggregation of myocilin hasten increased intraocular pressure and glaucoma-characteristic vision loss due to irreversible damage to the optic nerve. In spite of reports on the intracellular accumulation of mutant and WT myocilin in vitro, cell culture, and model organisms, these aggregates have not been structurally characterized. In this work, we provide biophysical evidence for the hallmarks of amyloid fibrils in aggregated forms of WT and mutant myocilin localized to the C-terminal olfactomedin (OLF) domain. These fibrils are grown under a variety of conditions in a nucleation-dependent and self-propagating manner. Protofibrillar oligomers and mature amyloid fibrils are observed in vitro. Full-length mutant myocilin expressed in mammalian cells forms intracellular amyloid-containing aggregates as well. Taken together, this work provides new insights into and raises new questions about the molecular properties of the highly conserved OLF domain, and suggests a novel protein-based hypothesis for glaucoma pathogenesis for further testing in a clinical setting.

The absence of Mertk in RCS rats results in defective RPE phagocytosis, accumulation of outer segment (OS) debris in the subretinal space, and subsequent death of photoreceptors. Previous research utilizing Mertk gene replacement therapy in RCS rats provided proof of concept for treatment of this form of recessive retinitis pigmentosa (RP); however, the beneficial effects on retinal function were transient. In the present study, we evaluated whether delivery of a MERTK transgene using a tyrosine-mutant AAV8 capsid could lead to more robust and longer-term therapeutic outcomes than previously reported.An AAV8 Y733F vector expressing a human MERTK cDNA driven by a RPE-selective promoter was administrated subretinally at postnatal day 2. Functional and morphological analyses were performed at 4 months and 8 months post-treatment. Retinal vasculature and Müller cell activation were analyzed by quantifying acellular capillaries and glial fibrillary acidic protein immunostaining, respectively.Electroretinographic responses from treated eyes were more than one-third of wild-type levels and OS were well preserved in the injection area even at 8 months. Rescue of RPE phagocytosis, prevention of retinal vasculature degeneration, and inhibition of Müller cell activation were demonstrated in the treated eyes for at least 8 months.This research describes a longer and much more robust functional and morphological rescue than previous studies. We also demonstrate for the first time that an AAV8 mutant capsid serotype vector has a substantial therapeutic potential for RPE-specific gene delivery. These results suggest that tyrosine-mutant AAV8 vectors hold promise for the treatment of individuals with MERTK-associated RP.

Recent studies indicate that mitochondrial proteins may contribute to the pathogenesis of primary open-angle glaucoma (POAG). In this study, we examined the association between POAG and common variations in gene-encoding mitochondrial proteins.We examined genetic data from 3430 POAG cases and 3108 controls derived from the combination of the GLAUGEN and NEIGHBOR studies. We constructed biological-system coherent mitochondrial nuclear-encoded protein gene-sets by intersecting the MitoCarta database with the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. We examined the mitochondrial gene-sets for association with POAG and with normal-tension glaucoma (NTG) and high-tension glaucoma (HTG) subsets using Pathway Analysis by Randomization Incorporating Structure.We identified 22 KEGG pathways with significant mitochondrial protein-encoding gene enrichment, belonging to six general biological classes. Among the pathway classes, mitochondrial lipid metabolism was associated with POAG overall (P = 0.013) and with NTG (P = 0.0006), and mitochondrial carbohydrate metabolism was associated with NTG (P = 0.030). Examining the individual KEGG pathway mitochondrial gene-sets, fatty acid elongation and synthesis and degradation of ketone bodies, both lipid metabolism pathways, were significantly associated with POAG (P = 0.005 and P = 0.002, respectively) and NTG (P = 0.0004 and P < 0.0001, respectively). Butanoate metabolism, a carbohydrate metabolism pathway, was significantly associated with POAG (P = 0.004), NTG (P = 0.001), and HTG (P = 0.010).We present an effective approach for assessing the contributions of mitochondrial genetic variation to open-angle glaucoma. Our findings support a role for mitochondria in POAG pathogenesis and specifically point to lipid and carbohydrate metabolism pathways as being important.

The electroretinogram (ERG) is a sensitive and noninvasive method for testing retinal function. In this protocol, we describe a method for performing ERGs in mice. Contact lenses on the mouse cornea measure the electrical response to a light stimulus of photoreceptors and downstream retinal cells, and the collected data are analyzed to evaluate retinal function.

Abstract Glaucoma is a multi-factorial blinding disease in which genetic factors play an important role. Elevated intraocular pressure is a highly heritable risk factor for primary open angle glaucoma and currently the only target for glaucoma therapy. Our study helps to better understand underlying genetic and molecular mechanisms that regulate intraocular pressure, and identifies a new candidate gene, Cacna2d1 , that modulates intraocular pressure and a promising therapeutic, pregabalin, which binds to CACNA2D1 protein and lowers intraocular pressure significantly. Because our study utilizes a genetically diverse population of mice with known sequence variants, we are able to determine that the intraocular pressure-lowering effect of pregabalin is dependent on the Cacna2d1 haplotype. Using human genome-wide association study (GWAS) data, evidence for association of a CACNA2D1 single-nucleotide polymorphism and primary open angle glaucoma is found. Importantly, these results demonstrate that our systems genetics approach represents an efficient method to identify genetic variation that can guide the selection of therapeutic targets.

Noncoding microRNAs (miRNAs) have been implicated in the pathogenesis of glaucoma. We aimed to identify common variants in miRNA coding genes (MIR) associated with primary open-angle glaucoma (POAG).Using the NEIGHBORHOOD data set (3853 cases/33,480 controls with European ancestry), we first assessed the relation between 85 variants in 76 MIR genes and overall POAG. Subtype-specific analyses were performed in high-tension glaucoma (HTG) and normal-tension glaucoma subsets. Second, we examined the expression of miR-182, which was associated with POAG, in postmortem human ocular tissues (ciliary body, cornea, retina, and trabecular meshwork [TM]), using miRNA sequencing (miRNA-Seq) and droplet digital PCR (ddPCR). Third, miR-182 expression was also examined in human aqueous humor (AH) by using miRNA-Seq. Fourth, exosomes secreted from primary human TM cells were examined for miR-182 expression by using miRNA-Seq. Fifth, using ddPCR we compared miR-182 expression in AH between five HTG cases and five controls.Only rs76481776 in MIR182 gene was associated with POAG after adjustment for multiple comparisons (odds ratio [OR] = 1.23, 95% confidence interval [CI]: 1.11-1.42, P = 0.0002). Subtype analysis indicated that the association was primarily in the HTG subset (OR = 1.26, 95% CI: 1.08-1.47, P = 0.004). The risk allele T has been associated with elevated miR-182 expression in vitro. Data from ddPCR and miRNA-Seq confirmed miR-182 expression in all examined ocular tissues and TM-derived exosomes. Interestingly, miR-182 expression in AH was 2-fold higher in HTG patients than nonglaucoma controls (P = 0.03) without controlling for medication treatment.Our integrative study is the first to associate rs76481776 with POAG via elevated miR-182 expression.

Retinal pigment epithelial (RPE) degeneration is potentially involved in the pathogenesis of several retinal degenerative diseases.mTORC1 signaling is shown as a crucial regulator of many biological processes and disease progression.In this study, we aimed at investigating the role of mTORC1 signaling in RPE degeneration.Methods: Western blots were conducted to detect mTORC1 expression pattern during RPE degeneration.Cre-loxP system was used to generate RPE-specific mTORC1 activation mice.Fundus, immunofluorescence staining, transmission electron microscopy, and targeted metabolomic analysis were conducted to determine the effects of mTORC1 activation on RPE degeneration in vivo.Electroretinography, spectral-domain optical coherence tomography, and histological experiments were conducted to determine the effects of mTORC1 activation on choroidal and retinal function in vivo.Results: RPE-specific activation of mTORC1 led to RPE degeneration as shown by the loss of RPE-specific marker, compromised cell junction integrity, and intracellular accumulation of lipid droplets.RPE degeneration further led to abnormal choroidal and retinal function.The inhibition of mTORC1 signaling with rapamycin could partially reverse RPE degeneration.Targeted metabolomics analysis further revealed that mTORC1 activation affected the metabolism of purine, carboxylic acid, and niacin in RPE. Conclusion:This study revealed that abnormal activation of mTORC1 signaling leads to RPE degeneration, which could provide a promising target for the treatment of RPE dysfunction-related diseases.

Mutations in the MERTK gene are responsible for retinal degeneration in the Royal College of Surgeons (RCS) rat and are a cause of human autosomal recessive retinitis pigmentosa (RP). This study reports the identification and functional analysis of novel MERTK mutations to provide information regarding whether they are causative of severe rod-cone degeneration in a young patient.MERTK missense variants identified by single-strand conformational polymorphism (SSCP) and sequence analysis were introduced into expression constructs and used to transfect HEK293T cells. Recombinant protein expression was assayed with anti-MERTK and anti-phosphotyrosine antibodies. Protein turnover was assayed in pulse-chase studies of 35S-methionine incorporation. Transcript levels were determined by quantitative RT-PCR.Three MERTK sequence variants were identified in a patient with rod-cone dystrophy: R722X in exon 16 and R865W in exon 19 on the paternal allele and R844C in exon 19 on the maternal allele. The R844C sequence change affects an evolutionarily conserved amino acid residue and was not detected in unaffected individuals. In transfected HEK293Tcells, wild-type (wt) and W865 MERTK were expressed at equivalent levels and present in the plasma membrane, stimulated tyrosine phosphorylation, and induced significant rounding of the cell bodies. In contrast, C844 MERTK was expressed at low levels and did not stimulate tyrosine phosphorylation. In addition, the relative stability of C844 MERTK was significantly less than wt in assays of protein turnover. At age 13, the patient had 20/60 and 20/200 acuities, tunnel vision of 5 degrees centrally, and a far temporal peripheral crescent bilaterally, and ERGs were nondetectable. The fundi showed bull's-eye macular atrophy and widespread RPE thinning.The present study reports the identification of R844C, the first putative pathogenic MERTK missense mutation that results in severe retinal degeneration with childhood onset when in compound heterozygous form with a R722X allele. The loss of function of C844 MERTK is probably due to decreased protein stability.

<b>Background:</b> Nail-patella syndrome (NPS) is a rare autosomal dominant syndrome, characterised by dysplasia of the nails, patellae, elbows and iliac horns. Mutations in the <i>LMX1B</i> gene were found in four North American families in whom glaucoma cosegregated with NPS. <b>Aims:</b> To investigate the association of glaucoma with NPS in Australian families and to determine how common NPS is in Australia. <b>Methods:</b> One family with NPS and glaucoma was identified from the Glaucoma Inheritance Study in Tasmania. A further 18 index cases of NPS were identified from the genetics database for southeastern Australia. Eight of these pedigrees were available for comprehensive glaucoma examination on available family members. DNA was sequenced for mutations in <i>LMX1B</i>. <b>Results:</b> In total, 52 living cases of NPS were identified suggesting a minimum prevalence of at least 1 in 100 000. 32 subjects from eight NPS pedigrees (four familial and four sporadic cases) were examined. 14 subjects had NPS alone. 4 subjects had NPS and glaucoma or ocular hypertension. Five pedigrees with NPS had a reported family history of glaucoma, although some of these people with glaucoma did not have NPS. <i>LMX1B</i> mutations were identified in 5 of the 8 index cases—three sporadic and two familial. Two of the six (33%) participants over 40 years of age had developed glaucoma, showing increased risk of glaucoma in NPS. <b>Conclusion:</b> Patients with NPS should be examined regularly for glaucoma. However, because the families with NPS are ascertained primarily from young probands or probands who are isolated cases, the exact level of risk is unclear.

Mutations in myocilin cause an inherited form of open angle glaucoma, a prevalent neurodegenerative disorder associated with increased intraocular pressure. Myocilin forms part of the trabecular meshwork extracellular matrix presumed to regulate intraocular pressure. Missense mutations, clustered in the olfactomedin (OLF) domain of myocilin, render the protein prone to aggregation in the endoplasmic reticulum of trabecular meshwork cells, causing cell dysfunction and death. Cellular studies have demonstrated temperature-sensitive secretion of myocilin mutants, but difficulties in expression and purification have precluded biophysical characterization of wild-type (wt) myocilin and disease-causing mutants in vitro. We have overcome these limitations by purifying wt and select glaucoma-causing mutant (D380A, I477N, I477S, K423E) forms of the OLF domain (228-504) fused to a maltose binding protein (MBP) from E. coli . Monomeric fusion proteins can be isolated in solution. To determine the relative stability of wt and mutant OLF domains, we developed a fluorescence thermal stability assay without removal of MBP and provide the first direct evidence that mutated OLF is folded but less thermally stable than wt. We tested the ability of seven chemical chaperones to stabilize mutant myocilin. Only sarcosine and trimethylamine N-oxide were capable of shifting the melting temperature of all mutants tested to near that of wt OLF. Our work lays the foundation for the identification of tailored small molecules capable of stabilizing mutant myocilin and promoting secretion to the extracellular matrix, to better control intraocular pressure and to ultimately delay the onset of myocilin glaucoma.

Open-angle glaucoma (OAG) is a major cause of blindness worldwide. To identify new risk loci for OAG, we performed a genome-wide association study in 3,071 OAG cases and 6,750 unscreened controls, and meta-analysed the results with GWAS data for intraocular pressure (IOP) and optic disc parameters (the overall meta-analysis sample size varying between 32,000 to 48,000 participants), which are glaucoma-related traits. We identified and independently validated four novel genome-wide significant associations within or near MYOF and CYP26A1, LINC02052 and CRYGS, LMX1B, and LMO7 using single variant tests, one additional locus (C9) using gene-based tests, and two genetic pathways - "response to fluid shear stress" and "abnormal retina morphology" - in pathway-based tests. Interestingly, some of the new risk loci contribute to risk of other genetically-correlated eye diseases including myopia and age-related macular degeneration. To our knowledge, this study is the first integrative study to combine genetic data from OAG and its correlated traits to identify new risk variants and genetic pathways, highlighting the future potential of combining genetic data from genetically-correlated eye traits for the purpose of gene discovery and mapping.

Genetic variants associated with primary open-angle glaucoma (POAG) are known to influence disease risk. However, the clinical effect of associated variants individually or in aggregate is not known. Genetic risk scores (GRS) examine the cumulative genetic load by combining individual genetic variants into a single measure, which is assumed to have a larger effect and increased power to detect relevant disease-related associations.To investigate if a GRS that comprised 12 POAG genetic risk variants is associated with age at disease diagnosis.A cross-sectional study included individuals with POAG and controls from the Glaucoma Genes and Environment (GLAUGEN) study and the National Eye Institute Glaucoma Human Genetics Collaboration (NEIGHBOR) study. A GRS was formulated using 12 variants known to be associated with POAG, and the alleles associated with increasing risk of POAG were aligned in the case-control sets. In case-only analyses, the association of the GRS with age at diagnosis was analyzed as an estimate of disease onset. Results from cohort-specific analyses were combined with meta-analysis. Data collection started in August 2012 for the NEIGHBOR cohort and in July 2008 for the GLAUGEN cohort and were analyzed starting in March 2018.Association of a 12 single-nucleotide polymorphism POAG GRS with age at diagnosis in individuals with POAG using linear regression.The GLAUGEN study included 976 individuals with POAG and 1140 controls. The NEIGHBOR study included 2132 individuals with POAG and 2290 controls. For individuals with POAG, the mean (SD) age at diagnosis was 63.6 (9.8) years in the GLAUGEN cohort and 66.0 (13.7) years in the NEIGHBOR cohort. For controls, the mean (SD) age at enrollment was 65.5 (9.2) years in the GLAUGEN cohort and 68.9 (11.4) years in the NEIGHBOR cohort. All study participants were European white. The GRS was strongly associated with POAG risk in case-control analysis (odds ratio per 1-point increase in score = 1.24; 95% CI, 1.21-1.27; P = 3.4 × 10-66). In case-only analyses, each higher GRS unit was associated with a 0.36-year earlier age at diagnosis (β = -0.36; 95% CI, -0.56 to -0.16; P = 4.0 × 10-4). Individuals in the top 5% of the GRS had a mean (SD) age at diagnosis of 5.2 (12.8) years earlier than those in the bottom 5% GRS (61.4 [12.7] vs 66.6 [12.9] years; P = 5.0 × 10-4).A higher dose of POAG risk alleles was associated with an earlier age at glaucoma diagnosis. On average, individuals with POAG with the highest GRS had 5.2-year earlier age at diagnosis of disease. These results suggest that a GRS that comprised genetic variants associated with POAG could help identify patients with risk of earlier disease onset impacting screening and therapeutic strategies.

A new avenue of mining published genome-wide association studies includes the joint analysis of related traits. The power of this approach depends on the genetic correlation of traits, which reflects the number of pleiotropic loci, i.e. genetic loci influencing multiple traits. Here, we applied new meta-analyses of optic nerve head (ONH) related traits implicated in primary open-angle glaucoma (POAG); intraocular pressure and central corneal thickness using Haplotype reference consortium imputations. We performed a multi-trait analysis of ONH parameters cup area, disc area and vertical cup-disc ratio. We uncover new variants; rs11158547 in PPP1R36-PLEKHG3 and rs1028727 near SERPINE3 at genome-wide significance that replicate in independent Asian cohorts imputed to 1000 Genomes. At this point, validation of these variants in POAG cohorts is hampered by the high degree of heterogeneity. Our results show that multi-trait analysis is a valid approach to identify novel pleiotropic variants for ONH.

To examine families ascertained for late-onset primary open-angle glaucoma (POAG) to determine mutations in the gene coding for myocilin.The diagnosis of late-onset POAG was defined as age at diagnosis more than 35 years, intraocular pressure (IOP) 22 mm Hg or more in both eyes or 19 mm Hg or more while the patient was taking two glaucoma medications, glaucomatous optic neuropathy in both eyes, and visual field loss consistent with optic nerve damage in at least one eye of the proband. Two of three criteria were required in other family members. DNA from all families was screened for polymorphisms in myocilin using single-strand conformation polymorphism analysis. All polymorphisms were sequenced for mutations.Eighty-three affected people in 29 families with late-onset POAG were screened for mutations. Three mutations, two novel missense (Thr377Met and Glu352Lys) and one nonsense (Gln368STOP), were identified. The missense mutations did not segregate with the disease phenotype in these families. The nonsense mutation was found in 3 of 29 unrelated families with POAG. All affected family members and 8 of 12 in whom glaucoma was suspected had the Gln368STOP mutation. All people with this mutation had elevated IOP, and 78% had POAG by age 70.Three mutations were identified in the gene coding for myocilin in families with late-onset POAG. Of these, the Gln368STOP mutation was highly associated with the development of glaucoma. All people with this mutation had glaucoma or elevated IOP by age 70. In the United States, the Gln368STOP mutation in myocilin is strongly associated with the development of late-onset POAG. However, factors in addition to the presence of this mutation seem to play a role in the development of ocular hypertension and glaucoma in these families.

Recent studies have demonstrated that glaucoma-causing mutant myocilin proteins are misfolded and retained in the endoplasmic reticulum of cells. We showed previously that P370L mutant myocilin is poorly secreted at 37 °C and prolonged expression of the protein in differentiated human trabecular meshwork cells results in abnormal morphology and cell killing. Culturing cells at a lower temperature, a condition known to facilitate protein folding, enhances secretion and reverses the cytotoxic effects. We wanted to determine if temperature sensitive secretion is a general property of myocilin missense mutants. Wild-type or mutant forms of myocilin were transiently expressed in HEK 293 cells cultured at either 37 or 30 °C and protein secretion was assessed by immunoblotting. Of 15 myocilin missense mutants tested, representing a range in severity of associated glaucoma phenotypes, 14 displayed increased secretion at 30 °C. The sole exception was K423E, which is associated with an unusual mode of glaucoma inheritance. Generally, there is an inverse relationship between the degree of mutant myocilin secretion at 30 °C and the severity of the associated glaucoma phenotype. Mutants that show abundant secretion at 30 °C such as T377M, G364V, I499F and D380A are associated with less virulent glaucoma phenotypes, while mutants such as P370L, I477N, and Y437H display little secretion at 30 °C and are associated with more virulent glaucoma phenotypes. We conclude that temperature sensitive secretion is a property of most olfactomedin-domain myocilin mutants. The correlation between temperature sensitive secretion and glaucoma phenotype likely reflects the intrinsic susceptibility to misfolding of individual mutant proteins. These results support the hypothesis that myocilin-induced glaucoma is a protein conformational disease. Facilitating mutant protein folding could be a new approach to development of therapies for this disease.

Primary open-angle glaucoma (POAG) is a leading cause of blindness worldwide. Using genome-wide association single-nucleotide polymorphism data from the Glaucoma Genes and Environment study and National Eye Institute Glaucoma Human Genetics Collaboration comprising 3,108 cases and 3,430 controls, we assessed biologic pathways as annotated in the KEGG database for association with risk of POAG. After correction for genic overlap among pathways, we found 4 pathways, butanoate metabolism (hsa00650), hematopoietic cell lineage (hsa04640), lysine degradation (hsa00310) and basal transcription factors (hsa03022) related to POAG with permuted p < 0.001. In addition, the human leukocyte antigen (HLA) gene family was significantly associated with POAG (p < 0.001). In the POAG subset with normal-pressure glaucoma (NPG), the butanoate metabolism pathway was also significantly associated (p < 0.001) as well as the MAPK and Hedgehog signaling pathways (hsa04010 and hsa04340), glycosaminoglycan biosynthesis-heparan sulfate pathway (hsa00534) and the phenylalanine, tyrosine and tryptophan biosynthesis pathway (hsa0400). The butanoate metabolism pathway overall, and specifically the aspects of the pathway that contribute to GABA and acetyl-CoA metabolism, was the only pathway significantly associated with both POAG and NPG. Collectively these results implicate GABA and acetyl-CoA metabolism in glaucoma pathogenesis, and suggest new potential therapeutic targets.

MERTK-associated retinal degenerations are thought to have defects in phagocytosis of shed outer segment membranes by the retinal pigment epithelium (RPE), as do the rodent models of these diseases. We have subretinally injected an RPE-specific AAV2 vector, AAV2-VMD2-hMERTK, to determine whether this would provide long-term photoreceptor rescue in the RCS rat, which it did for up to 6.5 months, the longest time point examined. Moreover, we found phagosomes in the RPE in the rescued regions of RCS retinas soon after the onset of light. The same vector also had a major protective effect in Mertk-null mice, with a concomitant increase in ERG response amplitudes in the vector-injected eyes. These findings suggest that planned clinical trials with this vector will have a favorable outcome.

Random segments of Myxococcus xanthus DNA were cloned in yeast artificial chromosomes (YACs) to construct a physical map of the genome. EcoRI restriction maps of 409 YAC clones with inserts averaging 111 kilobase pairs (kb) were determined. Comparison to the map of a 300-kb region of M. xanthus obtained from clones in Escherichia coli indicates that segments of DNA cloned in YACs are stably maintained in yeast and that their sequences accurately reflect the structure of the Myxococcus genome. The 409 YAC inserts were ordered within 60 map segments (contigs) by aligning their EcoRI restriction maps and by hybridization with 18 gene-specific DNA probes. These 60 map segments may represent the entire Myxococcus genome and could be used to organize its genetic information. This study illustrates the utility of YACs for cloning large segments of DNA and for reliable long-range genomic mapping.

Circulating estrogen levels are relevant in glaucoma phenotypic traits. We assessed the association between an estrogen metabolism single nucleotide polymorphism (SNP) panel in relation to primary open angle glaucoma (POAG), accounting for gender.We included 3,108 POAG cases and 3,430 controls of both genders from the Glaucoma Genes and Environment (GLAUGEN) study and the National Eye Institute Glaucoma Human Genetics Collaboration (NEIGHBOR) consortium genotyped on the Illumina 660W-Quad platform. We assessed the relation between the SNP panels representative of estrogen metabolism and POAG using pathway- and gene-based approaches with the Pathway Analysis by Randomization Incorporating Structure (PARIS) software. PARIS executes a permutation algorithm to assess statistical significance relative to the pathways and genes of comparable genetic architecture. These analyses were performed using the meta-analyzed results from the GLAUGEN and NEIGHBOR data sets. We evaluated POAG overall as well as two subtypes of POAG defined as intraocular pressure (IOP) ≥22 mmHg (high-pressure glaucoma [HPG]) or IOP <22 mmHg (normal pressure glaucoma [NPG]) at diagnosis. We conducted these analyses for each gender separately and then jointly in men and women.Among women, the estrogen SNP pathway was associated with POAG overall (permuted p=0.006) and HPG (permuted p<0.001) but not NPG (permuted p=0.09). Interestingly, there was no relation between the estrogen SNP pathway and POAG when men were considered alone (permuted p>0.99). Among women, gene-based analyses revealed that the catechol-O-methyltransferase gene showed strong associations with HTG (permuted gene p≤0.001) and NPG (permuted gene p=0.01).The estrogen SNP pathway was associated with POAG among women.

Abstract Retinitis pigmentosa (RP) shows progressive loss of photoreceptors involved with heterogeneous genetic background. Here, by exome sequencing and linkage analysis on a Chinese family with autosomal dominant RP, we identified a putative pathogenic variant, p.Gly97Arg, in the gene SPP2 , of which expression was detected in multiple tissues including retina. The p.Gly97Arg was absent in 800 ethnically matched chromosomes and 1400 in-house exome dataset and was located in the first of the two highly conserved disulfide bonded loop of secreted phosphoprotein 2 (Spp-24) encoded by SPP2 . Overexpression of p.Gly97Arg and another signal peptide mutation, p.Gly29Asp, caused cellular retention of both endogenous wild type and exogenous mutants in vitro and primarily affected rod photoreceptors in zebrafish mimicking cardinal feature of RP. Taken together, our data indicate that the two mutations of SPP2 have dominant negative effects and cellular accumulation of Spp-24 might be particularly toxic to photoreceptors and/or retinal pigment epithelium. SPP2 has a new role in retinal degeneration.

Abstract The retinal pigment epithelium (RPE) serves vital roles in ocular development and retinal homeostasis but has limited representation in large-scale functional genomics datasets. Understanding how common human genetic variants affect RPE gene expression could elucidate the sources of phenotypic variability in selected monogenic ocular diseases and pinpoint causal genes at genome-wide association study (GWAS) loci. We interrogated the genetics of gene expression of cultured human fetal RPE (fRPE) cells under two metabolic conditions and discovered hundreds of shared or condition-specific expression or splice quantitative trait loci (e/sQTLs). Co-localizations of fRPE e/sQTLs with age-related macular degeneration (AMD) and myopia GWAS data suggest new candidate genes, and mechanisms by which a common RDH5 allele contributes to both increased AMD risk and decreased myopia risk. Our study highlights the unique transcriptomic characteristics of fRPE and provides a resource to connect e/sQTLs in a critical ocular cell type to monogenic and complex eye disorders.

Purpose Genetic variants in regions that include the mitochondrial genes thioredoxin reductase 2 (TXNRD2) and malic enzyme 3 (ME3) are associated with primary open-angle glaucoma (POAG) in genome-wide association studies (GWASs). To assess their clinical impact, we investigated whether TXNRD2 and ME3 genetic risk scores (GRSs) are associated with specific glaucoma phenotypes. Design Cross-sectional study. Participants A total of 2617 patients with POAG and 2634 control participants from the National Eye Institute Glaucoma Human Genetics Collaboration Hereditable Overall Operational Database (NEIGHBORHOOD) consortium. Methods All POAG-associated single nucleotide polymorphisms (SNPs) in the TXNRD2 and ME3 loci were identified using GWAS data (P < 0.05). Of these, 20 TXNRD2 and 24 ME3 SNPs were selected after adjusting for linkage disequilibrium. The correlation between SNP effect size and gene expression levels was investigated using the Gene-Tissue Expression database. Genetic risk scores were constructed for each individual using the unweighted sum of TXNRD2, ME3, and TXNRD2 + ME3 combined risk alleles. Age- and sex-adjusted odds ratios (ORs) for POAG diagnosis were calculated per decile for each GRS. Additionally, the clinical features of patients with POAG in the top 1%, 5%, and 10% of each GRS were compared with those in the bottom 1%, 5%, and 10%, respectively. Main Outcome Measures Primary open-angle glaucoma OR per GRS decile, maximum treated intraocular pressure (IOP), and prevalence of paracentral visual field loss among patients with POAG with high versus low GRSs. Results A larger SNP effect size strongly correlated with higher TXNRD2 and lower ME3 expression levels (r = 0.95 and r = –0.97, respectively; P < 0.05 for both). Individuals in decile 10 of the TXNRD2 + ME3 GRS had the highest odds of POAG diagnosis (OR, 1.79 compared with decile 1; 95% confidence interval, 1.39–2.30; P < 0.001). Patients with POAG in the top 1% of the TXNRD2 GRS showed higher mean maximum treated IOP compared with the bottom 1% (19.9 mmHg vs. 15.6 mmHg; adjusted P = 0.03). Patients with POAG in the top 1% of the ME3 and TXNRD2 + ME3 GRS showed a higher prevalence of paracentral field loss than the bottom 1% (72.7% vs. 14.3% for ME3 GRS and 88.9% vs. 33.3% for TXNRD2+ME3 GRS; adjusted P = 0.03 for both). Conclusions Patients with POAG with higher TXNRD2 and ME3 GRSs showed higher treated IOP and a greater prevalence of paracentral field loss. Functional studies exploring how these variants impact mitochondrial function in patients with glaucoma are warranted. Financial Disclosure(s) Proprietary or commercial disclosure may be found after the references. Genetic variants in regions that include the mitochondrial genes thioredoxin reductase 2 (TXNRD2) and malic enzyme 3 (ME3) are associated with primary open-angle glaucoma (POAG) in genome-wide association studies (GWASs). To assess their clinical impact, we investigated whether TXNRD2 and ME3 genetic risk scores (GRSs) are associated with specific glaucoma phenotypes. Cross-sectional study. A total of 2617 patients with POAG and 2634 control participants from the National Eye Institute Glaucoma Human Genetics Collaboration Hereditable Overall Operational Database (NEIGHBORHOOD) consortium. All POAG-associated single nucleotide polymorphisms (SNPs) in the TXNRD2 and ME3 loci were identified using GWAS data (P < 0.05). Of these, 20 TXNRD2 and 24 ME3 SNPs were selected after adjusting for linkage disequilibrium. The correlation between SNP effect size and gene expression levels was investigated using the Gene-Tissue Expression database. Genetic risk scores were constructed for each individual using the unweighted sum of TXNRD2, ME3, and TXNRD2 + ME3 combined risk alleles. Age- and sex-adjusted odds ratios (ORs) for POAG diagnosis were calculated per decile for each GRS. Additionally, the clinical features of patients with POAG in the top 1%, 5%, and 10% of each GRS were compared with those in the bottom 1%, 5%, and 10%, respectively. Primary open-angle glaucoma OR per GRS decile, maximum treated intraocular pressure (IOP), and prevalence of paracentral visual field loss among patients with POAG with high versus low GRSs. A larger SNP effect size strongly correlated with higher TXNRD2 and lower ME3 expression levels (r = 0.95 and r = –0.97, respectively; P < 0.05 for both). Individuals in decile 10 of the TXNRD2 + ME3 GRS had the highest odds of POAG diagnosis (OR, 1.79 compared with decile 1; 95% confidence interval, 1.39–2.30; P < 0.001). Patients with POAG in the top 1% of the TXNRD2 GRS showed higher mean maximum treated IOP compared with the bottom 1% (19.9 mmHg vs. 15.6 mmHg; adjusted P = 0.03). Patients with POAG in the top 1% of the ME3 and TXNRD2 + ME3 GRS showed a higher prevalence of paracentral field loss than the bottom 1% (72.7% vs. 14.3% for ME3 GRS and 88.9% vs. 33.3% for TXNRD2+ME3 GRS; adjusted P = 0.03 for both). Patients with POAG with higher TXNRD2 and ME3 GRSs showed higher treated IOP and a greater prevalence of paracentral field loss. Functional studies exploring how these variants impact mitochondrial function in patients with glaucoma are warranted.

Abstract Although glaucoma is a disease modulated by eye pressure, the mechanisms of pressure sensing in the eye are not well understood. Here, we investigated associations between mechanosensitive ion channel gene variants and primary open-angle glaucoma (POAG). Common (minor allele frequency &gt; 5%) single nucleotide polymorphisms located within the genomic regions of 20 mechanosensitive ion channel genes in the K2P, TMEM63, PIEZO and TRP channel families were assessed using genotype data from the NEIGHBORHOOD consortium of 3853 cases and 33,480 controls. Rare (minor allele frequency &lt; 1%) coding variants were assessed using exome array genotyping data for 2606 cases and 2606 controls. Association with POAG was analyzed using logistic regression adjusting for age and sex. Two rare PIEZO1 coding variants with protective effects were identified in the NEIGHBOR dataset: R1527H, (OR 0.17, P = 0.0018) and a variant that alters a canonical splice donor site, g.16-88737727-C-G Hg38 (OR 0.38, P = 0.02). Both variants showed similar effects in the UK Biobank and the R1527H also in the FinnGen database. Several common variants also reached study-specific thresholds for association in the NEIGHBORHOOD dataset. These results identify novel variants in several mechanosensitive channel genes that show associations with POAG, suggesting that these channels may be potential therapeutic targets.

Vision loss degrades the quality of life of aged individuals. The major cause in industrialized countries is age-related macular degeneration (AMD), a blinding eye disease due to death of photoreceptors in the macula, a specialized retinal region responsible for high acuity vision. Photoreceptor death in AMD is thought to follow damage to the retinal pigment epithelium (RPE) [1], a monolayer of polarized, post-mitotic cells located between the photoreceptors and the choroidal blood supply that performs a variety of crucial tasks [2]. One proposed mechanism of RPE dysfunction in AMD posits a lifetime of oxidative damage leading to deposits (termed drusen) between the RPE and choroid, inflammation [3], and diminished RPE mitochondrial function [4, 5]. Macular RPE mitochondrial DNA from AMD eyes is more damaged than corresponding macular nuclear DNA [6], and macular RPE mitochondrial DNA damage correlates positively with AMD severity [7]. To model RPE mitochondrial DNA damage in AMD, we selectively ablated mitochondrial DNA replication and transcription in the RPE of postnatal mice [8]. The resulting deficit in RPE oxidative phosphorylation (OXPHOS) caused a slowly progressive photoreceptor degeneration, as well as a number of RPE morphological changes similar to those seen in AMD. The most prominent early RPE changes were hypertrophy and dedifferentiation, which coincided with activation of the mTOR pathway in OXPHOS-deficient RPE cells. Robust mTOR activation in the context of OXPHOS deficiency is counterintuitive because mTOR integrates trophic factor and nutrient availability signals to regulate cell growth and proliferation [9], and poisoning of mitochondrial energy production inhibits mTOR [10]. The fact that ATP levels in OXPHOS-deficient RPE cells were not substantially different from controls helps to resolve this apparent paradox. Levels of selected glycolytic metabolites were increased by several orders of magnitude, indicating a large glycolytic flux capable of generating ATP at a high rate. However, dependence on aerobic glycolysis is not a requirement for mTOR activation; acute treatment of wild-type mice with a strong oxidant that the targets the RPE also activated mTOR and triggered dedifferentiation, with profound negative consequences for adjacent photoreceptors [8]. Features suggestive of RPE hypertrophy and/or dedifferentiation have been reported for a number of other mouse retinal degeneration models [11-13], suggesting that a mTOR-associated RPE stress response may be quite general. OXPHOS deficiency leads eventually to RPE atrophy, which is seen more commonly in AMD than RPE hypertrophy. Our findings suggest that RPE hypertrophy may be present at earlier stages of AMD. Indeed, ocular coherence tomography imaging demonstrated thickened macular RPE more frequently in early AMD eyes than in advanced AMD or control eyes (C. Zhao, unpublished). RPE hypertrophy may be less prominent in advanced AMD because drusen and diminished transport through aged Bruch's basement membrane [14] may restrict access of RPE cells to nutrients from the choroidal blood supply. RPE cells in most mouse models are presumably not limited in this regard, facilitating mTOR activation. Hence, the stress response we have identified may shed light on RPE-related disease processes in which nutrients are readily available. Intriguingly, pharmacological inhibition of mTORC1 with rapamycin blunted RPE dedifferentiation and hypertrophy and preserved photoreceptor numbers and function for both the metabolic and oxidative stress models [8]. Rapamycin has recently been shown to have the remarkable ability to increase the longevity of mice, even when administered late in life [15]. Our results thus connect age-dependent retinal degeneration with a pathway known to be critical for the determination of lifespan. An in depth understanding is needed of the requirements for mTOR activation in the RPE and the mechanism by which the pathway mediates RPE dedifferentiation, with the goal of combating age-related retinal disease and extending human healthspan.

Purpose: The accumulation of undigestible autofluorescent material (UAM), termed lipofuscin in vivo, is a hallmark of aged RPE. Lipofuscin derives, in part, from the incomplete degradation of phagocytized photoreceptor outer segments (OS). Whether this accumulated waste is toxic is unclear. We therefore investigated the effects of UAM in highly differentiated human fetal RPE (hfRPE) cultures. Methods: Unmodified and photo-oxidized OS were fed daily to confluent cultures of ARPE-19 RPE or hfRPE. The emission spectrum, composition, and morphology of resulting UAM were measured and compared to in vivo lipofuscin. Effects of UAM on multiple RPE phenotypes were assessed. Results: Compared to ARPE-19, hfRPE were markedly less susceptible to UAM buildup. Accumulated UAM in hfRPE initially resembled the morphology of lipofuscin from AMD eyes, but compacted and shifted spectrum over time to resemble lipofuscin from healthy aged human RPE. UAM accumulation mildly reduced transepithelial electrical resistance, ketogenesis, certain RPE differentiation markers, and phagocytosis efficiency, while inducing senescence and rare, focal pockets of epithelial-mesenchymal transition. However, it had no effects on mitochondrial oxygen consumption rate, certain other RPE differentiation markers, secretion of drusen components or polarity markers, nor cell death. Conclusions: hfRPE demonstrates a remarkable resistance to UAM accumulation, suggesting mechanisms for efficient OS processing that may be lost in other RPE culture models. Furthermore, while UAM alters hfRPE phenotype, the effects are modest, consistent with conflicting reports in the literature on the toxicity of lipofuscin. Our results suggest that healthy RPE may adequately adapt to and tolerate lipofuscin accumulation.

Purpose To describe the phenotype and genotype of a family with suspected Sorsby fundus dystrophy (SFD). Design Case reports and results of deoxyribonucleic acid (DNA) analysis. Methods Clinical features were determined by complete ophthalmologic examination or by review of medical records. Mutational analysis of the tissue inhibitor of metalloproteinase (TIMP)3 gene was performed by DNA resequencing. Biochemical properties of the mutant TIMP3 protein were studied, and phylogenetic and molecular modeling analyses of TIMP proteins were performed. Results Fundi of four affected family members demonstrated active or regressed bilateral choroidal neovascularization, whereas another affected individual displayed severe diffuse pigmentary degeneration associated with nyctalopia characteristic of SFD. Onset of disease occurred in the fifth to seventh decades of life. A heterozygous His158Arg mutation was found in seven affected family members and was absent from an unaffected member and 98 unrelated controls. Bioinformatic analyses indicate that histidine 158 is an evolutionarily conserved residue in most vertebrate TIMP homologs and predict that substitution by arginine disrupts TIMP3 function. The mutant protein appears to be expressed by fibroblasts from an affected family member. Molecular modeling suggests that TIMP3 residue 158 may be part of a protein-protein interaction interface. Conclusion A novel mutation in TIMP3 causes a late-onset form of SFD in this family. His158Arg is the first reported TIMP3 SFD coding sequence mutation that does not create an unpaired cysteine. Further study of this unusual mutation may provide insight into the mechanism of SFD pathogenesis. To describe the phenotype and genotype of a family with suspected Sorsby fundus dystrophy (SFD). Case reports and results of deoxyribonucleic acid (DNA) analysis. Clinical features were determined by complete ophthalmologic examination or by review of medical records. Mutational analysis of the tissue inhibitor of metalloproteinase (TIMP)3 gene was performed by DNA resequencing. Biochemical properties of the mutant TIMP3 protein were studied, and phylogenetic and molecular modeling analyses of TIMP proteins were performed. Fundi of four affected family members demonstrated active or regressed bilateral choroidal neovascularization, whereas another affected individual displayed severe diffuse pigmentary degeneration associated with nyctalopia characteristic of SFD. Onset of disease occurred in the fifth to seventh decades of life. A heterozygous His158Arg mutation was found in seven affected family members and was absent from an unaffected member and 98 unrelated controls. Bioinformatic analyses indicate that histidine 158 is an evolutionarily conserved residue in most vertebrate TIMP homologs and predict that substitution by arginine disrupts TIMP3 function. The mutant protein appears to be expressed by fibroblasts from an affected family member. Molecular modeling suggests that TIMP3 residue 158 may be part of a protein-protein interaction interface. A novel mutation in TIMP3 causes a late-onset form of SFD in this family. His158Arg is the first reported TIMP3 SFD coding sequence mutation that does not create an unpaired cysteine. Further study of this unusual mutation may provide insight into the mechanism of SFD pathogenesis.

Spermatogenesis is a highly specialized cell differentiation process regulated by the testicular microenvironment. During the process of spermatogenesis, phagocytosis performs an essential role in male germ cell development, and its dysfunction in the testis can cause reproduction defects. MerTK, as a critical protein of phagocytosis, facilitates the removal of apoptotic substrates from the retina and ovaries through cooperation with several phagocytosis receptors. However, its role in mammalian spermatogenesis remains undefined. Here, we found that 30-week-old MerTK−/− male mice developed oligoasthenospermia due to abnormal spermatogenesis. These mice showed damaged seminiferous tubule structure, as well as altered spermatogonia proliferation and differentiation. We also found that Sertoli cells from MerTK−/− mice had decreased phagocytic activity on apoptotic germ cells in vitro. Moreover, a transcriptomic analysis demonstrated that the pivotal genes involved in spermatid differentiation and development changed expression. These results indicate that MerTK is crucial for spermatogenesis, as it regulates the crosstalk between germ cells and Sertoli cells. This provides us insight into the molecular mechanism of MerTK on spermatogenesis and its implications for the diagnosis and treatment of human male infertility.

Primary open-angle glaucoma (POAG) is the most common chronic optic neuropathy worldwide. Epidemiological studies show a robust positive relation between intraocular pressure (IOP) and POAG and modest positive association between IOP and blood pressure (BP), while the relation between BP and POAG is controversial. The International Glaucoma Genetics Consortium (n=27 558), the International Consortium on Blood Pressure (n=69 395), and the National Eye Institute Glaucoma Human Genetics Collaboration Heritable Overall Operational Database (n=37 333), represent genome-wide data sets for IOP, BP traits and POAG, respectively. We formed genome-wide significant variant panels for IOP and diastolic BP and found a strong relation with POAG (odds ratio and 95% confidence interval: 1.18 (1.14-1.21), P=1.8 × 10-27) for the former trait but no association for the latter (P=0.93). Next, we used linkage disequilibrium (LD) score regression, to provide genome-wide estimates of correlation between traits without the need for additional phenotyping. We also compared our genome-wide estimate of heritability between IOP and BP to an estimate based solely on direct measures of these traits in the Erasmus Rucphen Family (ERF; n=2519) study using Sequential Oligogenic Linkage Analysis Routines (SOLAR). LD score regression revealed high genetic correlation between IOP and POAG (48.5%, P=2.1 × 10-5); however, genetic correlation between IOP and diastolic BP (P=0.86) and between diastolic BP and POAG (P=0.42) were negligible. Using SOLAR in the ERF study, we confirmed the minimal heritability between IOP and diastolic BP (P=0.63). Overall, IOP shares genetic basis with POAG, whereas BP has limited shared genetic correlation with IOP or POAG.

Greg Lemke’s laboratory was one of the pioneers of research into the TAM family of receptor tyrosine kinases (RTKs). Not only was Tyro3 cloned in his laboratory, but his group also extensively studied mice knocked out for individual or various combinations of the TAM RTKs Tyro3, Axl, and Mertk. Here we primarily focus on one of the paralogs—MERTK. We provide a historical perspective on rodent models of loss of Mertk function and their association with retinal degeneration and blindness. We describe later studies employing mouse genetics and the generation of newer knockout models that point out incongruencies with the inference that loss of MERTK-dependent phagocytosis is sufficient for severe, early-onset photoreceptor degeneration in mice. This discussion is meant to raise awareness with regards to the limitations of the original Mertk knockout mouse model generated using 129 derived embryonic stem cells and carrying 129 derived alleles and the role of these alleles in modifying Mertk knockout phenotypes or even displaying Mertk-independent phenotypes. We also suggest molecular approaches that can further Greg Lemke’s scintillating legacy of dissecting the molecular functions of MERTK—a protein that has been described to function in phagocytosis as well as in the negative regulation of inflammation.

Sperm typing was used to measure recombination fractions among pseudoautosomal markers and the beginning of the X/Y-specific sequences located at the pseudoautosomal boundary. These experiments included primer-extension preamplification and PCR followed by allele typing using gel electrophoresis. A newly developed data-analysis program allowed the construction of the first multipoint-linkage sperm-typing map, using results obtained on seven loci from three individuals. The large sample size not only confirmed the increased recombination activity of the pseudoautosomal region but allowed an estimate of interference of recombination to be made. The coefficient of coincidence was calculated to be .26 over a physical distance of only approximately 1,800 kb. The observation of a few sperm presumably resulting from double recombination argues that more than one crossover event can occur in this region during male meiosis.

Vascular perfusion may be impaired in primary open-angle glaucoma (POAG); thus, we evaluated a panel of markers in vascular tone-regulating genes in relation to POAG.We used Illumina 660W-Quad array genotype data and pooled P-values from 3108 POAG cases and 3430 controls from the combined National Eye Institute Glaucoma Human Genetics Collaboration consortium and Glaucoma Genes and Environment studies. Using information from previous literature and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, we compiled single-nucleotide polymorphisms (SNPs) in 186 vascular tone-regulating genes. We used the 'Pathway Analysis by Randomization Incorporating Structure' analysis software, which performed 1000 permutations to compare the overall pathway and selected genes with comparable randomly generated pathways and genes in their association with POAG.The vascular tone pathway was not associated with POAG overall or POAG subtypes, defined by the type of visual field loss (early paracentral loss (n=224 cases) or only peripheral loss (n=993 cases)) (permuted P≥0.20). In gene-based analyses, eight were associated with POAG overall at permuted P<0.001: PRKAA1, CAV1, ITPR3, EDNRB, GNB2, DNM2, HFE, and MYL9. Notably, six of these eight (the first six listed) code for factors involved in the endothelial nitric oxide synthase activity, and three of these six (CAV1, ITPR3, and EDNRB) were also associated with early paracentral loss at P<0.001, whereas none of the six genes reached P<0.001 for peripheral loss only.Although the assembled vascular tone SNP set was not associated with POAG, genes that code for local factors involved in setting vascular tone were associated with POAG.

To evaluate the performance of N-[2-(diethylamino)ethyl]-(18)F-5-fluoropicolinamide ((18)F-P3BZA) for visualizing porcine retinal pigment epithelium (pRPE) cells transplanted in the striatum for the treatment of Parkinson disease and to monitor the long-term activity of implanted pRPE cells by means of (18)F-P3BZA positron emission tomography (PET)/computed tomography (CT) in vivo.Animal work was conducted in accordance with the administrative panel on laboratory animal care. In vitro cell uptake of (18)F-P3BZA was determined with incubation of melanotic pRPE or amelanotic ARPE-19 cells with (18)F-P3BZA. To visualize the implanted pRPE cells in vivo, normal rats (four per group) were injected with pRPE or ARPE-19 cells attached to gelatin microcarriers in the left striatum and with control gelatin microcarriers in the right striatum and followed up with small animal PET/CT. Longitudinal PET/CT scans were acquired in 12 rats up to 16 days after surgery. Postmortem analysis, which included autoradiography and hematoxylin-eosin, Fontana-Masson, and immunofluorescence staining, was performed. Data were compared with the Student t test, analysis of variance, and regression analysis.(18)F-P3BZA accumulated in pRPE cells effectively (3.48% of the injected dose [ID] per gram of brain tissue ± 0.58 at 1 hour after injection of the probe at 2 days after surgery in vivo) but not in control ARPE-19 cells (P < .05). Longitudinal PET/CT scans revealed that the activity of implanted pRPE cells decreased over time, as evidenced by a reduction in (18)F-P3BZA uptake (3.39% ID/g ± 0.18, 2.49% ID/g ± 0.41, and 1.20% ID/g ± 0.13 at days 2, 9, and 16, respectively; P < .05). Postmortem analysis helped confirm the results of in vivo imaging.(18)F-P3BZA PET/CT is a feasible technique for visualizing and detecting the activity of implanted RPE cells in vivo.

Sex hormones may be associated with primary open-angle glaucoma (POAG), although the mechanisms are unclear. We previously observed that gene variants involved with estrogen metabolism were collectively associated with POAG in women but not men; here we assessed gene variants related to testosterone metabolism collectively and POAG risk.We used two datasets: one from the United States (3853 cases and 33,480 controls) and another from Australia (1155 cases and 1992 controls). Both datasets contained densely called genotypes imputed to the 1000 Genomes reference panel. We used pathway- and gene-based approaches with Pathway Analysis by Randomization Incorporating Structure (PARIS) software to assess the overall association between a panel of single nucleotide polymorphisms (SNPs) in testosterone metabolism genes and POAG. In sex-stratified analyses, we evaluated POAG overall and POAG subtypes defined by maximum IOP (high-tension [HTG] or normal tension glaucoma [NTG]).In the US dataset, the SNP panel was not associated with POAG (permuted P = 0.77), although there was an association in the Australian sample (permuted P = 0.018). In both datasets, the SNP panel was associated with POAG in men (permuted P ≤ 0.033) and not women (permuted P ≥ 0.42), but in gene-based analyses, there was no consistency on the main genes responsible for these findings. In both datasets, the testosterone pathway association with HTG was significant (permuted P ≤ 0.011), but again, gene-based analyses showed no consistent driver gene associations.Collectively, testosterone metabolism pathway SNPs were consistently associated with the high-tension subtype of POAG in two datasets.

Abstract We investigated the effects of treating differentiated retinal pigment epithelial (RPE) cells with didanosine (ddI), which is associated with retinopathy in individuals with HIV/AIDS. We hypothesized that such treatment would cause depletion of mitochondrial DNA and provide insight into the consequences of degradation of RPE mitochondrial function in aging and disease. Treatment of differentiated ARPE-19 or human primary RPE cells with 200 µM ddI for 6–24 days was not cytotoxic but caused up to 60% depletion of mitochondrial DNA, and a similar reduction in mitochondrial membrane potential and NDUFA9 protein abundance. Mitochondrial DNA-depleted RPE cells demonstrated enhanced aerobic glycolysis by extracellular flux analysis, increased AMP kinase activation, reduced mTOR activity, and increased resistance to cell death in response to treatment with the oxidant, sodium iodate. We conclude that ddI-mediated mitochondrial DNA depletion promotes a glycolytic shift in differentiated RPE cells and enhances resistance to oxidative damage. Our use of ddI treatment to induce progressive depletion of mitochondrial DNA in differentiated human RPE cells should be widely applicable for other studies aimed at understanding RPE mitochondrial dysfunction in aging and disease.

The selective silencing of target genes in specific cell types by RNA interference (RNAi) represents a powerful approach both to gene therapy of dominantly active mutant alleles, and to the investigation of normal gene function in animal models in vivo. We established a simple and versatile in vitro method for screening the efficacy of DNA-based short hairpin RNAs (shRNAs), and identified a highly effective shRNA targeting basic fibroblast growth factor (bFGF), a gene thought to play important roles in endogenous neuroprotective responses in the rat retina. We used two viral vectors, based on lentivirus and adeno-associated virus (AAV), to deliver shRNAs and silence bFGF in retinal pigment epithelial cells in vivo. The AAV experiments made use of a "stabilized double-stranded" version of these vectors with rapid onset of gene expression. In the rat retinal pigment epithelium, shRNAs delivered by either vector reduced bFGF immunoreactivity to undetectable levels in transduced cells, whereas a nonfunctional control construct incorporating a two-base pair mutation had no measurable effect on bFGF expression. Silencing commenced within a few days after injection of virus and remained stable throughout the period of observation, as long as 60 days. Viral delivery of RNAi constructs offers a powerful and versatile approach for both gene therapy and the analysis of fundamental questions in retinal biology.

Purpose.: To determine the basis and to characterize the phenotype of a chemically induced mutation in a mouse model of retinal degeneration. Methods.: Screening by indirect ophthalmoscopy identified a line of N-ethyl-N-nitrosourea (ENU) mutagenized mice demonstrating retinal patches. Longitudinal studies of retinal histologic sections showed photoreceptors in the peripheral retina undergoing slow, progressive degeneration. The mutation was named neuroscience mutagenesis facility 12 (nmf12), and mapping localized the critical region to Chromosome 2. Results.: Sequencing of nmf12 DNA revealed a point mutation in the c-mer tyrosine kinase gene, designated Mertknmf12 . We detected elevated levels of tumor necrosis factor (Tnf, previously Tnfa) in retinas of Mertknmf12 homozygotes relative to wild-type controls and investigated whether the increase of TNF, an inflammatory cytokine produced by macrophages/monocytes that signals intracellularly to cause necrosis or apoptosis, could underlie the retinal degeneration observed in Mertknmf12 homozygotes. Mertknmf12 homozygous mice were mated to mice lacking the entire Tnf gene and partial coding sequences of the Lta (Tnfb) and Ltb (Tnfc) genes. 2 B6.129P2-Ltb/Tnf/Ltatm1Dvk /J homozygotes did not exhibit a retinal degeneration phenotype and will, hereafter, be referred to as Tnfabc −/− mice. Surprisingly, mice homozygous for both the Mertknmf12 and the Ltb/Tnf/Ltatm1Dvk allele (Tnfabc −/−) demonstrated an increase in the rate of retinal degeneration. Conclusions.: These findings illustrate that a mutation in the Mertk gene leads to a significantly slower progressive retinal degeneration compared with other alleles of Mertk. These results demonstrate that TNF family members play a role in protecting photoreceptors of Mertknmf12 homozygotes from cell death.

Abnormalities of the retinal pigment epithelial (RPE) cells are seen in several inherited degenerations such as Best disease (Petrukhin et al., 1998; Marmorstein et al., 2000), Stargardt disease (Weng et al., 1999), Sorsby’s fundus dystrophy (Steinmetz et al., 1992; Jacobson et al., 1995), and childhood-onset severe retinal dystrophy (Gu et al., 1997) and Leber congenital amaurosis due to RPE65 mutations (Marlhens et al., 1997; Morimura et al., 1998; Lotery et al., 2000; Thompson et al., 2000). RCS rats have been studied extensively as a model of photoreceptor (PR)-RPE cell interactions (Mullen and LaVail, 1976; LaVail, 2001) and retinal degeneration (Strauss et al., 1998; LaVail, 2001). Retinal degeneration in RCS results from failure of the RPE to phagocytize shed rod outer segments (OS) (Herron et al., 1969; Bok and Hall, 1971). The unphagocytized OS membranes form membranous whorls at the RPE surface, and the rod OS grow abnormally long (Dowling and Sidman, 1962; LaVail and Battelle, 1975). Eventually, the OS layer degenerates into a debris zone with subsequent PR cell death (Dowling and Sidman, 1962).

Abstract Several neurotrophic factors (NTFs) are effective in protecting retinal photoreceptor cells from the damaging effects of constant light and slowing the rate of inherited photoreceptor degenerations. It is currently unclear whether, if continuously available, all NTFs can be protective for many or most retinal degenerations (RDs). We used transgenic mice that continuously overexpress the neurotrophin NT‐3 from lens fibers under the control of the αA‐crystallin promoter to test for neuroprotection in light‐damage experiments and in four naturally occurring or transgenically induced RDs in mice. Lens‐specific expression of NT‐3 mRNA was demonstrated both by in situ hybridization in embryos and by reverse‐transcriptase polymerase chain reaction (RT‐PCR) in adult mice. Furthermore, NT‐3 protein was found in abundance in the lens, ocular fluids, and retina by enzyme‐linked immunosorbent assay (ELISA) and immunocytochemistry. Overexpression of NT‐3 had no adverse effects on the structure or function of the retina for up to at least 14 months of age. Mice expressing the NT‐3 transgene were protected from the damaging effects of constant light to a much greater degree than those receiving bolus injections of NT‐3. When the NT‐3 transgene was transferred into rd/rd, Rds /+, Q344ter mutant rhodopsin or Mertk knockout mice, overexpression of NT‐3 had no protective effect on the RDs in these mice. Thus, specificity of the neuroprotective effect of NT‐3 is clearly demonstrated, and different molecular mechanisms are inferred to mediate the protective effect in light‐induced and inherited RDs. J. Comp. Neurol. 511:724–735, 2008. © 2008 Wiley‐Liss, Inc.

Mitochondrial diseases are a clinically heterogenous group of disorders caused by respiratory chain dysfunction and associated with progressive, multi-systemic phenotype. There is no effective treatment or cure, and no FDA-approved drug for treating mitochondrial disease. To identify and characterize potential therapeutic compounds, we developed an in vitro screening assay and identified a group of direct AMP-activated protein kinase (AMPK) activators originally developed for the treatment of diabetes and metabolic syndrome. Unlike previously investigated AMPK agonists such as AICAR, these compounds allosterically activate AMPK in an AMP-independent manner, thereby increasing specificity and decreasing pleiotropic effects. The direct AMPK activator PT1 significantly improved mitochondrial function in assays of cellular respiration, energy status, and cellular redox. PT1 also protected against retinal degeneration in a mouse model of photoreceptor degeneration associated with mitochondrial dysfunction and oxidative stress, further supporting the therapeutic potential of AMP-independent AMPK agonists in the treatment of mitochondrial disease.

Retinal pigment epithelium (RPE) cell migration in response to disease has been reported for age-related macular degeneration, proliferative vitreoretinopathy, and proliferative diabetic retinopathy. The complex molecular process of RPE cell migration is regulated in part by growth factors and cytokines, and activation of the PI3/AKT/mTOR signaling pathway. Rapamycin, an allosteric mTOR inhibitor, has been shown to block only one of the primary downstream mTOR effectors, p70 S6 kinase 1, in many cell types. INK128, a selective mTOR ATP binding site competitor, blocks both p70 S6 kinase 1 and a second primary downstream effector, 4E-BP1. We performed scratch assays using differentiated ARPE-19 and primary porcine RPE cells to assess the effect of mTOR inhibition on cell migration. We found that INK128-mediated blocking of both p70 S6 kinase 1 and 4E-BP1 was much more effective at preventing RPE cell migration than rapamycin-mediated inhibition of p70 S6 kinase 1 alone.

The BEST1 gene has been implicated in distinguishable ocular diseases ranging from macular degeneration to nanophthalmos.1Marmorstein A.D. Cross H.E. Peachey N.S. Functional roles of bestrophins in ocular epithelia.Prog Retin Eye Res. 2009; 28: 206-226Crossref PubMed Scopus (105) Google Scholar Homozygous BEST1 mutations were previously associated with several diseases1Marmorstein A.D. Cross H.E. Peachey N.S. Functional roles of bestrophins in ocular epithelia.Prog Retin Eye Res. 2009; 28: 206-226Crossref PubMed Scopus (105) Google Scholar including autosomal-recessive bestrophinopathy (ARB), which was characterized by macular degeneration with scattered punctate deposits and accumulation of fluid within and/or beneath the neurosensory retina.2Burgess R. Millar I.D. Leroy B.P. et al.Biallelic mutation of BEST1 causes a distinct retinopathy in humans.Am J Hum Genet. 2008; 82: 19-31Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar The significant clinical heterogeneity associated with BEST1 mutations requires an extensive genetic analysis and comprehensive clinical characterization of patients to better understand genotype–phenotype correlations.We studied a consanguineous Chinese family (Fig 1A, available at http://aaojournal.org) with 5 individuals possessing complex ocular phenotypes. Complete ophthalmic examinations were performed on all family members with the exception of patient II:5, who participated only in our genetic study. All human studies were approved by the local institutional ethical review board in accordance with the Declaration of Helsinki.Individuals I:1 and I:2 are first-degree cousins and had no history of eye diseases at any point in their lives. Four patients (II:6, II:12, II:13, and III:1) manifested angle-closure glaucoma (ACG) as demonstrated by darkroom gonioscopy, ultrasound biomicroscopy, increased intraocular pressure, and increased cup-to-disc ratio. Consistent with ACG, all 4 patients had moderately shortened axial lengths with mild hyperopia. They also had bilateral cortical cataracts, which by patient report developed before the age of 20 years (Table 1 and Fig 2, available at http://aaojournal.org).Fundus examination revealed in all 4 patients characteristic features of ARB including retinal pigment epithelium (RPE) disturbances and scattered punctate yellow-whitish fleck deposits surrounding maculae, and accumulation of fluid in the subretinal and/or intraretinal space at the fovea (Table 1 and Fig 2). Moreover, a significant reduction in the EOG light rise with moderately decreased ERG responses was detected in all patients, implying a primary insult to the RPE.2Burgess R. Millar I.D. Leroy B.P. et al.Biallelic mutation of BEST1 causes a distinct retinopathy in humans.Am J Hum Genet. 2008; 82: 19-31Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar Patient II:5 was unavailable for our clinical reevaluation, but her medical records indicated the development of bilateral ACG, premature cataract and maculopathy by 40 years of age. A total of 13 other family members were examined and had no remarkable ophthalmic findings.To detect the genetic causes of the complicated phenotypes observed in this family, we employed a previously described targeted next-generation sequencing approach to screen 179 genes associated with hereditary retinal dystrophies for mutations3Chen X.J. Zhao K.X. Sheng X.L. et al.Targeted sequencing of 179 genes associated with hereditary retinal dystrophies and 10 candidate genes identifies novel and known mutations in patients with various retinal diseases.Invest Ophthalmol Vis Sci. 2013; 54: 2186-2197Crossref PubMed Scopus (62) Google Scholar in the proband (II:13). A total of 2298 single nucleotide variations and 78 indels were initially detected by next-generation sequencing and, after bioinformatic filtering, 12 variants were validated by Sanger sequencing and evaluated for pathogenicity as previously described.3Chen X.J. Zhao K.X. Sheng X.L. et al.Targeted sequencing of 179 genes associated with hereditary retinal dystrophies and 10 candidate genes identifies novel and known mutations in patients with various retinal diseases.Invest Ophthalmol Vis Sci. 2013; 54: 2186-2197Crossref PubMed Scopus (62) Google Scholar By this process, only 1 variant, c.752G>A (p.C251Y) in BEST1, was identified as a putative pathogenic mutation. Sanger sequencing confirmed the presence of this variant in all 5 patients in the homozygous state, and in 10 unaffected family members in the heterozygous state (Fig 1A). Of note, patient III:1 is homozygous for the mutation, suggesting that his unaffected father, who is not known to be related to the family, also harbors this variant, presumably owing to a founder effect. This variant is absent in the 3 remaining unaffected family members tested and in 100 unrelated controls, is evolutionarily conserved (Fig 1B) and is predicted to be functionally highly deleterious by the bioinformatic programs PolyPhen-2 (score = 1) and SIFT (score = 0).Because ACG and cataract are very rarely reported in families with ARB, we considered the possibility that these additional phenotypes in affected family members were associated with mutations in other genes genetically linked to BEST1. To localize such candidate genes, we performed homozygosity mapping across all of chromosome 11 using 20 microsatellite markers and 3 single nucleotide polymorphisms. The homozygous region shared by all patients is flanked by D11S4109 and rs7130122 (Fig 1A), and as expected, includes BEST1. We next analyzed the genes within the homozygous region using the OMIM online database (http://omim.org/) and found only 2 genes, TMEM138 and TMEM216, which may be associated with ocular anomalies. Sanger sequencing of the coding regions of these genes did not detect any putative pathogenic mutations. Thus, homozygosity for the p.C251Y mutation of BEST1 is very likely the cause of the entire spectrum of phenotypes observed in the family.The mechanism by which p.C251Y causes the phenotypes we have described is unknown. Bestrophin-1 encoded by BEST1 is a RPE protein hypothesized to function as a Ca2+ activated Cl− channel, or a regulator of ion transport.1Marmorstein A.D. Cross H.E. Peachey N.S. Functional roles of bestrophins in ocular epithelia.Prog Retin Eye Res. 2009; 28: 206-226Crossref PubMed Scopus (105) Google Scholar Previously described biallelic BEST1 mutations associated with ARB are probably loss-of-function mutations.2Burgess R. Millar I.D. Leroy B.P. et al.Biallelic mutation of BEST1 causes a distinct retinopathy in humans.Am J Hum Genet. 2008; 82: 19-31Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar, 4Pomares E. Bures-Jelstrup A. Ruiz-Nogales S. et al.Nonsense-mediated decay as the molecular cause for autosomal recessive bestrophinopathy in two unrelated families.Invest Ophthalmol Vis Sci. 2012; 53: 532-537Crossref PubMed Scopus (24) Google Scholar p.C251Y is located in the fifth transmembrane spanning helix (TM5),5Milenkovic V.M. Rivera A. Horling F. Weber B.H. Insertion and topology of normal and mutant bestrophin-1 in the endoplasmic reticulum membrane.J Biol Chem. 2007; 282: 1313-1321Crossref PubMed Scopus (69) Google Scholar very close to the extracellular surface (Fig 1C). Interestingly, all mutants previously found in TM5 and TM6, 2 symmetric helices, caused dominant phenotypes (Fig 1C). These dominant-acting mutations were hypothesized to generate unclear dysfunction rather than loss of BEST1 function.1Marmorstein A.D. Cross H.E. Peachey N.S. Functional roles of bestrophins in ocular epithelia.Prog Retin Eye Res. 2009; 28: 206-226Crossref PubMed Scopus (105) Google Scholar p.C251Y may have complex pathogenic effects including both loss-of-function and dysfunction, leading to more complicated phenotypes than other known ARB-associated alleles.ACG and hyperopia were previously reported in a small fraction of patients with ARB,2Burgess R. Millar I.D. Leroy B.P. et al.Biallelic mutation of BEST1 causes a distinct retinopathy in humans.Am J Hum Genet. 2008; 82: 19-31Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar but were not recognized as traits associated with ARB owing to their low frequency and lack of sufficient familial evidence. Patients with dominant MRCS syndrome (microcornea, retinal dystrophy, cataract, and posterior staphyloma) caused by heterozygous BEST1 mutations can develop premature cataracts, but ours is the first description of which we are aware of cataracts consistently found in patients with ARB. In this relatively large consanguineous family with recessive disease, the cosegregation of p.C251Y with a coherent ocular phenotype in all 5 patients strongly suggests a new constellation of traits associated with BEST1 homozygous mutations: ARB, ACG, hyperopia, and cataracts. Our extensive genetic analyses likely rule out other genetic components in this family. The BEST1 gene has been implicated in distinguishable ocular diseases ranging from macular degeneration to nanophthalmos.1Marmorstein A.D. Cross H.E. Peachey N.S. Functional roles of bestrophins in ocular epithelia.Prog Retin Eye Res. 2009; 28: 206-226Crossref PubMed Scopus (105) Google Scholar Homozygous BEST1 mutations were previously associated with several diseases1Marmorstein A.D. Cross H.E. Peachey N.S. Functional roles of bestrophins in ocular epithelia.Prog Retin Eye Res. 2009; 28: 206-226Crossref PubMed Scopus (105) Google Scholar including autosomal-recessive bestrophinopathy (ARB), which was characterized by macular degeneration with scattered punctate deposits and accumulation of fluid within and/or beneath the neurosensory retina.2Burgess R. Millar I.D. Leroy B.P. et al.Biallelic mutation of BEST1 causes a distinct retinopathy in humans.Am J Hum Genet. 2008; 82: 19-31Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar The significant clinical heterogeneity associated with BEST1 mutations requires an extensive genetic analysis and comprehensive clinical characterization of patients to better understand genotype–phenotype correlations. We studied a consanguineous Chinese family (Fig 1A, available at http://aaojournal.org) with 5 individuals possessing complex ocular phenotypes. Complete ophthalmic examinations were performed on all family members with the exception of patient II:5, who participated only in our genetic study. All human studies were approved by the local institutional ethical review board in accordance with the Declaration of Helsinki. Individuals I:1 and I:2 are first-degree cousins and had no history of eye diseases at any point in their lives. Four patients (II:6, II:12, II:13, and III:1) manifested angle-closure glaucoma (ACG) as demonstrated by darkroom gonioscopy, ultrasound biomicroscopy, increased intraocular pressure, and increased cup-to-disc ratio. Consistent with ACG, all 4 patients had moderately shortened axial lengths with mild hyperopia. They also had bilateral cortical cataracts, which by patient report developed before the age of 20 years (Table 1 and Fig 2, available at http://aaojournal.org). Fundus examination revealed in all 4 patients characteristic features of ARB including retinal pigment epithelium (RPE) disturbances and scattered punctate yellow-whitish fleck deposits surrounding maculae, and accumulation of fluid in the subretinal and/or intraretinal space at the fovea (Table 1 and Fig 2). Moreover, a significant reduction in the EOG light rise with moderately decreased ERG responses was detected in all patients, implying a primary insult to the RPE.2Burgess R. Millar I.D. Leroy B.P. et al.Biallelic mutation of BEST1 causes a distinct retinopathy in humans.Am J Hum Genet. 2008; 82: 19-31Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar Patient II:5 was unavailable for our clinical reevaluation, but her medical records indicated the development of bilateral ACG, premature cataract and maculopathy by 40 years of age. A total of 13 other family members were examined and had no remarkable ophthalmic findings. To detect the genetic causes of the complicated phenotypes observed in this family, we employed a previously described targeted next-generation sequencing approach to screen 179 genes associated with hereditary retinal dystrophies for mutations3Chen X.J. Zhao K.X. Sheng X.L. et al.Targeted sequencing of 179 genes associated with hereditary retinal dystrophies and 10 candidate genes identifies novel and known mutations in patients with various retinal diseases.Invest Ophthalmol Vis Sci. 2013; 54: 2186-2197Crossref PubMed Scopus (62) Google Scholar in the proband (II:13). A total of 2298 single nucleotide variations and 78 indels were initially detected by next-generation sequencing and, after bioinformatic filtering, 12 variants were validated by Sanger sequencing and evaluated for pathogenicity as previously described.3Chen X.J. Zhao K.X. Sheng X.L. et al.Targeted sequencing of 179 genes associated with hereditary retinal dystrophies and 10 candidate genes identifies novel and known mutations in patients with various retinal diseases.Invest Ophthalmol Vis Sci. 2013; 54: 2186-2197Crossref PubMed Scopus (62) Google Scholar By this process, only 1 variant, c.752G>A (p.C251Y) in BEST1, was identified as a putative pathogenic mutation. Sanger sequencing confirmed the presence of this variant in all 5 patients in the homozygous state, and in 10 unaffected family members in the heterozygous state (Fig 1A). Of note, patient III:1 is homozygous for the mutation, suggesting that his unaffected father, who is not known to be related to the family, also harbors this variant, presumably owing to a founder effect. This variant is absent in the 3 remaining unaffected family members tested and in 100 unrelated controls, is evolutionarily conserved (Fig 1B) and is predicted to be functionally highly deleterious by the bioinformatic programs PolyPhen-2 (score = 1) and SIFT (score = 0). Because ACG and cataract are very rarely reported in families with ARB, we considered the possibility that these additional phenotypes in affected family members were associated with mutations in other genes genetically linked to BEST1. To localize such candidate genes, we performed homozygosity mapping across all of chromosome 11 using 20 microsatellite markers and 3 single nucleotide polymorphisms. The homozygous region shared by all patients is flanked by D11S4109 and rs7130122 (Fig 1A), and as expected, includes BEST1. We next analyzed the genes within the homozygous region using the OMIM online database (http://omim.org/) and found only 2 genes, TMEM138 and TMEM216, which may be associated with ocular anomalies. Sanger sequencing of the coding regions of these genes did not detect any putative pathogenic mutations. Thus, homozygosity for the p.C251Y mutation of BEST1 is very likely the cause of the entire spectrum of phenotypes observed in the family. The mechanism by which p.C251Y causes the phenotypes we have described is unknown. Bestrophin-1 encoded by BEST1 is a RPE protein hypothesized to function as a Ca2+ activated Cl− channel, or a regulator of ion transport.1Marmorstein A.D. Cross H.E. Peachey N.S. Functional roles of bestrophins in ocular epithelia.Prog Retin Eye Res. 2009; 28: 206-226Crossref PubMed Scopus (105) Google Scholar Previously described biallelic BEST1 mutations associated with ARB are probably loss-of-function mutations.2Burgess R. Millar I.D. Leroy B.P. et al.Biallelic mutation of BEST1 causes a distinct retinopathy in humans.Am J Hum Genet. 2008; 82: 19-31Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar, 4Pomares E. Bures-Jelstrup A. Ruiz-Nogales S. et al.Nonsense-mediated decay as the molecular cause for autosomal recessive bestrophinopathy in two unrelated families.Invest Ophthalmol Vis Sci. 2012; 53: 532-537Crossref PubMed Scopus (24) Google Scholar p.C251Y is located in the fifth transmembrane spanning helix (TM5),5Milenkovic V.M. Rivera A. Horling F. Weber B.H. Insertion and topology of normal and mutant bestrophin-1 in the endoplasmic reticulum membrane.J Biol Chem. 2007; 282: 1313-1321Crossref PubMed Scopus (69) Google Scholar very close to the extracellular surface (Fig 1C). Interestingly, all mutants previously found in TM5 and TM6, 2 symmetric helices, caused dominant phenotypes (Fig 1C). These dominant-acting mutations were hypothesized to generate unclear dysfunction rather than loss of BEST1 function.1Marmorstein A.D. Cross H.E. Peachey N.S. Functional roles of bestrophins in ocular epithelia.Prog Retin Eye Res. 2009; 28: 206-226Crossref PubMed Scopus (105) Google Scholar p.C251Y may have complex pathogenic effects including both loss-of-function and dysfunction, leading to more complicated phenotypes than other known ARB-associated alleles. ACG and hyperopia were previously reported in a small fraction of patients with ARB,2Burgess R. Millar I.D. Leroy B.P. et al.Biallelic mutation of BEST1 causes a distinct retinopathy in humans.Am J Hum Genet. 2008; 82: 19-31Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar but were not recognized as traits associated with ARB owing to their low frequency and lack of sufficient familial evidence. Patients with dominant MRCS syndrome (microcornea, retinal dystrophy, cataract, and posterior staphyloma) caused by heterozygous BEST1 mutations can develop premature cataracts, but ours is the first description of which we are aware of cataracts consistently found in patients with ARB. In this relatively large consanguineous family with recessive disease, the cosegregation of p.C251Y with a coherent ocular phenotype in all 5 patients strongly suggests a new constellation of traits associated with BEST1 homozygous mutations: ARB, ACG, hyperopia, and cataracts. Our extensive genetic analyses likely rule out other genetic components in this family. Supplementary dataTable 1Clinical Features of 4 Patients in Family J90Patient IDGenderAge (yrs)BCVARefraction (D)IOP⁎The IOP refers to the value measured at the first visit. (mm Hg)ACD (mm)GonioscopyAL (mm)CataractCDRFundusERGEOGII:6M50OD: 6/60OS: CFOD: +3.50OS: +3.00OD: 13.0OS: 38.6OD: 1.65OS: 1.64OU:AC > 270°OD: 21.18OS: 21.29Bilateral, developed before 20 year of ageOD: 0.5OS: 0.8OU: Yellowish fleck deposits and RPE atrophy in maculae, SRF at foveaReduced rod and cone ERG by ∼50%Nearly absent light riseII:12F38OD: LPOS: 6/60OD: +1.75OS: +1.50OD: 34.5OS: 25.2OD: 1.45OS: 1.41OU:AC > 270°OD: 21.44OS: 21.30Bilateral, developed before 20 year of ageOD: 0.9OS: 0.9OU: Yellowish fleck deposits at maculae, macular edemaNAReduced light rise by ∼90%II:13M35OD: 6/40OS: 6/60OD: +2.75OS: +3.50OD: 13.8OS: 44.1OD: 1.59OS: 1.62OU:AC > 270°OD: 21.34OS: 20.89Bilateral, developed before 20 year of ageOD: 0.6 OS: 0.9OU: Macular edema, yellowish deposits and RPE atrophy in the maculae and midperipheryReduced rod and cone ERG by ∼50%Reduced light rise by ∼80%III:1M33OD: 6/60OS: 6/60OD: +1.75OS: +2.50OD: 22.2 OS: 19.7OD: 2.43OS: 2.06OD: AC > 180°OS: AC < 90°OD: 21.54OS: 21.36Bilateral, developed before 20 years of ageOD: 0.6OS: 0.3OU: Yellowish fleck deposits and RPE atrophy in maculae, macular edemaReduced rod and cone ERG by ∼50%Reduced light rise by ∼80%ACD = anterior chamber depth; AC = angle closure; AL = axial length; BCVA = best-corrected visual acuity; CDR = cup-to-disc ratio; CF = counting fingers within 30 cm; IOP = intraocular pressure; LP = light perception; NA = not available; OD = right eye; OS = left eye; OU = both eyes; SRF = subretinal fluid; VL = vitelliform lesion. The IOP refers to the value measured at the first visit. Open table in a new tab Figure 2Clinical evaluations of 2 patients, II:13 (upper left) and II:6 (upper right). A–D, Ultrasound biomicroscopy revealed shallow anterior chamber and angle-closure. E–H, Color funduscopy indicated macular degeneration with yellowish-white deposits (black arrows), and retinal pigment epithelium (RPE) pigmentary atrophy (white asterisk) in both patients. Optic disc pale with increased cup-to-disc ratio (CDR) were observed in both eyes of II:13 (E, F) and left eye of II:6 (H). I–L, Optical coherence tomography (OCT) examination demonstrated dramatic bilateral macular cystoid edema of II:13 (I, J), and moderate accumulation of fluid in the subretinal space with RPE detachment at the fovea in II:6 (K, L). White arrows indicate fluid accumulation.View Large Image Figure ViewerDownload Hi-res image Download (PPT) ACD = anterior chamber depth; AC = angle closure; AL = axial length; BCVA = best-corrected visual acuity; CDR = cup-to-disc ratio; CF = counting fingers within 30 cm; IOP = intraocular pressure; LP = light perception; NA = not available; OD = right eye; OS = left eye; OU = both eyes; SRF = subretinal fluid; VL = vitelliform lesion.

Abstract Objective: Several attributes of female reproductive history, including age at natural menopause (ANM), have been related to primary open-angle glaucoma (POAG). We assembled 18 previously reported common genetic variants that predict ANM to determine their association with ANM or POAG. Methods: Using data from the Nurses’ Health Study (7,143 women), we validated the ANM weighted genetic risk score in relation to self-reported ANM. Subsequently, to assess the relation with POAG, we used data from 2,160 female POAG cases and 29,110 controls in the National Eye Institute Glaucoma Human Genetics Collaboration Heritable Overall Operational Database (NEIGHBORHOOD), which consists of 8 datasets with imputed genotypes to 5.6+ million markers. Associations with POAG were assessed in each dataset, and site-specific results were meta-analyzed using the inverse weighted variance method. Results: The genetic risk score was associated with self-reported ANM ( P = 2.2 × 10 –77 ) and predicted 4.8% of the variance in ANM. The ANM genetic risk score was not associated with POAG (Odds Ratio (OR) = 1.002; 95% Confidence Interval (CI): 0.998, 1.007; P = 0.28). No single genetic variant in the panel achieved nominal association with POAG ( P ≥0.20). Compared to the middle 80 percent, there was also no association with the lowest 10 th percentile or highest 90 th percentile of genetic risk score with POAG (OR = 0.75; 95% CI: 0.47, 1.21; P = 0.23 and OR = 1.10; 95% CI: 0.72, 1.69; P = 0.65, respectively). Conclusions: A genetic risk score predicting 4.8% of ANM variation was not related to POAG; thus, genetic determinants of ANM are unlikely to explain the previously reported association between the two phenotypes.

To help determine whether there is a genetic basis to the substantial variability observed in nail-patella syndrome (NPS), we devised a scoring system that quantifies the severity of the orthopaedic characteristics in NPS. Use of this system to score affected members in three generations of a single kindred revealed wide variability of severity of orthopaedic findings both within and between generations. Genetic testing in this family supported, but did not prove, a previously reported theory that the severity of the NPS in the offspring is modulated by the allele contributed by the unaffected parent. Evaluation of nonorthopaedic characteristics revealed the presence of glaucoma and the absence of kidney disease in this family. It is important that patients with NPS be evaluated for renal disease and glaucoma.

The Seahorse XFp platform is widely used for metabolic assessment of cultured cells. Current methods require replating of cells into specialized plates. This is problematic for certain cell types, such as primary human fetal RPE (hfRPE) cells, which must be cultured for months to become properly differentiated. Our goal was to overcome this limitation by devising a method for assaying intact cell monolayers with the Seahorse XFp, without the need for replating.Primary hfRPE cells were differentiated by prolonged culture on filter inserts. Triangular sections of filters with differentiated cells attached were excised, transferred to XFp cell culture miniplate wells, immobilized at the bottoms, and subjected to mitochondrial stress tests. Replated cells were measured for comparison. Differentiated hfRPE cells were challenged or not with bovine photoreceptor outer segments (POS), and mitochondrial stress tests were performed 3.5 h later, after filter excision and transfer to assay plates.Differentiated hfRPE cells assayed following filter excision demonstrated increased maximal respiration, increased spare respiration capacity, and increased extracellular acidification rate (ECAR) relative to replated controls. hfRPE cells challenged with POS exhibited increased maximal respiration and spare capacity, with no apparent change in the ECAR, relative to untreated controls.We have developed a method to reproducibly assay intact, polarized monolayers of hfRPE cells with the Seahorse XFp platform and have shown that the method yields more robust metabolic measurements compared to standard methods and is suitable for assessing the consequences of prolonged perturbations of differentiated cells. We expect our approach to be useful for a variety of studies involving metabolic assessment of adherent cells cultured on filters.

Cre-mediated modulation of gene function in the murine retinal pigment epithelium (RPE) has been widely used, but current postnatal RPE-selective Cre driver lines suffer from limited recombination efficiency and/or ectopic or mosaic expression. We sought to generate a transgenic mouse line with consistently efficient RPE-selective Cre activity that could be temporally regulated. We used ϕC31 integrase to insert a DNA construct encoding a human BEST1 promoter fragment driving a Cre recombinase estrogen receptor fusion (BEST1-CreERT2) at the Rosa26 locus of C57BL/6J mice. Rosa26BEST1-CreERT2 mice were bred with a tdTomato reporter line and to mice with a Cre-conditional allele of Tfam. 4-hydroxytamoxifen or vehicle was delivered by four consecutive daily intraperitoneal injections. TdTomato was robustly expressed in the RPE of mice of both sexes for inductions beginning at P14 (males 90.7 ± 4.5%, females 84.7 ± 3.2%) and at 7 weeks (males 84.3 ± 7.0%, females 82 ± 3.6%). <0.6% of Muller glia also expressed tdTomato, but no tdTomato fluorescence was observed in other ocular cells or in multiple non-ocular tissues, with the exception of sparse foci in the testis. No evidence of retinal toxicity was observed in mice homozygous for the transgene induced beginning at P14 and assessed at 7-10 months. RPE-selective ablation of Tfam beginning at P14 led to reduced retinal thickness at 8 months of age and diminished retinal electrical responses at 12 months, as expected. These findings demonstrate that we have generated a mouse line with consistently efficient, tamoxifen-mediated postnatal induction of Cre recombination in the RPE and a small fraction of Muller glia. This line should be useful for temporally regulated modulation of gene function in the murine RPE.

Journal Article Four PCR-based polymorphisms in the pseudoautosomal region of the human X and Y chromosomes Get access Karin Schmitt, Karin Schmitt Search for other works by this author on: Oxford Academic PubMed Google Scholar Douglas Vollrath, Douglas Vollrath 1Howard Hughes Research Laboratories at Whitehead Institute, Massachusetts Institute of TechnologyCambridge, MA 02142, USA Search for other works by this author on: Oxford Academic PubMed Google Scholar Simon Foote, Simon Foote 1Howard Hughes Research Laboratories at Whitehead Institute, Massachusetts Institute of TechnologyCambridge, MA 02142, USA Search for other works by this author on: Oxford Academic PubMed Google Scholar Elizabeth M.C. Fisher, Elizabeth M.C. Fisher 1Howard Hughes Research Laboratories at Whitehead Institute, Massachusetts Institute of TechnologyCambridge, MA 02142, USA Search for other works by this author on: Oxford Academic PubMed Google Scholar David C. Page, David C. Page 1Howard Hughes Research Laboratories at Whitehead Institute, Massachusetts Institute of TechnologyCambridge, MA 02142, USA Search for other works by this author on: Oxford Academic PubMed Google Scholar Norman Amheim Norman Amheim * * To whom correspondence should be addressed Search for other works by this author on: Oxford Academic PubMed Google Scholar Human Molecular Genetics, Volume 2, Issue 11, November 1993, Page 1978, https://doi.org/10.1093/hmg/2.11.1978 Published: 01 November 1993

Contour-clamped homogeneous electric field (CHEF) gelelectrophoresis is a particular formulation of pulsed-field gel electrophoresis (PFGE), which uses an array of electrodes positioned around the gel (on a contour) and clamped to specific voltages to produce a nearly homogeneous electric field inside the contour (1). The direction of the electric field is changed periodically, as with all pulsed-field techniques. In the case of CHEF, field reorientation is achieved electronically by changing the voltages (potentials) of the various electrodes in the array (see Fig. 1). Commercial CHEF devices currently employ a hexagonal electrode array, but other types of contours, such as circles or squares, if properly clamped, can also produce alternating homogeneous electric fields.

Abstract The eye is an intricate organ with limited representation in large-scale functional genomics datasets. The retinal pigment epithelium (RPE) serves vital roles in ocular development and retinal homeostasis. We interrogated the genetics of gene expression of cultured human fetal RPE (fRPE) cells under two metabolic conditions. Genes with disproportionately high fRPE expression are enriched for genes related to inherited ocular diseases. Variants near these fRPE-selective genes explain a larger fraction of risk for both age-related macular degeneration (AMD) and myopia than variants near genes enriched in 53 other human tissues. Increased mitochondrial oxidation of glutamine by fRPE promoted expression of lipid synthesis genes implicated in AMD. Expression and splice quantitative trait loci (e/sQTL) analysis revealed shared and metabolic condition-specific loci of each type and several eQTL not previously described in any tissue. Fine mapping of fRPE e/sQTL across AMD and myopia genome-wide association data suggests new candidate genes, and mechanisms by which the same common variant of RDH5 contributes to both increased AMD risk and decreased myopia risk. Our study highlights the unique transcriptomic characteristics of fRPE and provides a resource to connect e/sQTL in a critical ocular cell type to monogenic and complex eye disorders.

Human chromosomal region 1q24 encodes two cloned disease genes and lies within large genetic inclusion intervals for several disease genes that have yet to be identified. We have constructed a single bacterial artificial chromosome (BAC) clone contig that spans over 2 Mb of 1q24 and consists of 78 clones connected by 100 STSs. The average density of mapped STSs is one of the highest described for a multimegabase region of the human genome. The contig was efficiently constructed by generating STSs from clone ends, followed by library walking. Distance information was added by determining the insert sizes of all clones, and expressed sequence tags (ESTs) and genes were incorporated to create a partial transcript map of the region, providing candidate genes for local disease loci. The gene order and content of the region provide insight into ancient duplication events that have occurred on proximal 1q. The stage is now set for further elucidation of this interesting region through large-scale sequencing.

Abstract Primary cilia are conserved organelles that integrate extracellular cues into intracellular signals and are critical for diverse processes, including cellular development and repair responses. Deficits in ciliary function cause multisystemic human diseases known as ciliopathies. In the eye, atrophy of the retinal pigment epithelium (RPE) is a common feature of many ciliopathies. However, the roles of RPE cilia in vivo remain poorly understood. In this study, we first found that mouse RPE cells only transiently form primary cilia. We then examined the RPE in the mouse model of Bardet-Biedl Syndrome 4 (BBS4), a ciliopathy associated with retinal degeneration in humans, and found that ciliation in BBS4 mutant RPE cells is disrupted early during development. Next, using a laser-induced injury model in vivo, we found that primary cilia in RPE reassemble in response to laser injury during RPE wound healing and then rapidly disassemble after the repair is completed. Finally, we demonstrated that RPE-specific depletion of primary cilia in a conditional mouse model of cilia loss promoted wound healing and enhanced cell proliferation. In summary, our data suggest that RPE cilia contribute to both retinal development and repair and provide insights into potential therapeutic targets for more common RPE degenerative diseases.

Summary To help determine whether there is a genetic basis to the substantial variability observed in nail–patella syndrome (NPS), we devised a scoring system that quantifies the severity of the orthopaedic characteristics in NPS. Use of this system to score affected members in three generations of a single kindred revealed wide variability of severity of orthopaedic findings both within and between generations. Genetic testing in this family supported, but did not prove, a previously reported theory that the severity of the NPS in the offspring is modulated by the allele contributed by the unaffected parent. Evaluation of nonorthopaedic characteristics revealed the presence of glaucoma and the absence of kidney disease in this family. It is important that patients with NPS be evaluated for renal disease and glaucoma.