Multimodal Imaging-Guided Stem Cell Ocular Treatment.

Stem cell therapies are gaining traction as promising treatments for a variety of degenerative conditions. Both clinical and preclinical studies of regenerative medicine are hampered by the lack of technologies that can evaluate the migration and behavior of stem cells post-transplantation. This study proposes an innovative method to longitudinally image in vivo human-induced pluripotent stem cells differentiated to retinal pigment epithelium (hiPSC-RPE) cells by multimodal photoacoustic microscopy, optical coherence tomography, and fluorescence imaging powered by ultraminiature chain-like gold nanoparticle cluster (GNC) nanosensors. The GNC exhibits an optical absorption peak in the near-infrared regime, and the 7-8 nm size in diameter after disassembly enables renal excretion and improved safety as well as biocompatibility. In a clinically relevant rabbit model, GNC-labeled hiPSC-RPE cells migrated to RPE degeneration areas and regenerated damaged tissues. The hiPSC-RPE cells' distribution and migration were noninvasively, longitudinally monitored for 6 months with exceptional sensitivity and spatial resolution. This advanced platform for cellular imaging has the potential to enhance regenerative cell-based therapies.

Oxidative stress differentially impacts apical and basolateral secretion of angiogenic factors from human iPSC-derived retinal pigment epithelium cells.

The retinal pigment epithelium (RPE) is a polarized monolayer that secretes growth factors and cytokines towards the retina apically and the choroid basolaterally. Numerous RPE secreted proteins have been linked to the pathogenesis of age-related macular degeneration (AMD). The purpose of this study was to determine the differential apical and basolateral secretome of RPE cells, and the effects of oxidative stress on directional secretion of proteins linked to AMD and angiogenesis. Tandem mass tag spectrometry was used to profile proteins in human iPSC-RPE apical and basolateral conditioned media. Changes in secretion after oxidative stress induced by H2O2 or tert-butyl hydroperoxide (tBH) were investigated by ELISA and western analysis. Out of 926 differentially secreted proteins, 890 (96%) were more apical. Oxidative stress altered the secretion of multiple factors implicated in AMD and neovascularization and promoted a pro-angiogenic microenvironment by increasing the secretion of pro-angiogenic molecules (VEGF, PTN, and CRYAB) and decreasing the secretion of anti-angiogenic molecules (PEDF and CFH). Apical secretion was impacted more than basolateral for PEDF, CRYAB and CFH, while basolateral secretion was impacted more for VEGF, which may have implications for choroidal neovascularization. This study lays a foundation for investigations of dysfunctional RPE polarized protein secretion in AMD and other chorioretinal degenerative disorders.

X-Chromosome Inactivation Is a Biomarker of Clinical Severity in Female Carriers of RPGR-Associated X-Linked Retinitis Pigmentosa.

PURPOSE: X-linked retinitis pigmentosa can manifest in female carriers with widely variable severity, whereas others remain unaffected. The contribution of X-chromosome inactivation (XCI) to phenotypic variation has been postulated but not demonstrated. Furthermore, the impact of genotype and genetic modifiers has been demonstrated in affected males but has not been well established in female carriers. The purpose of this study was to describe the scope of clinical phenotype in female carriers with mutations in RPGR and quantify the contribution of genotype, genetic modifiers, and XCI to phenotypic severity. DESIGN: Cohort study. PARTICIPANTS: Seventy-seven female carriers with RPGR mutations from 41 pedigrees. METHODS: Coding single nucleotide polymorphisms were sequenced in candidate genetic modifier genes encoding known RPGR-interacting proteins. X-chromosome inactivation ratios were determined in genomic DNA isolated from blood (n = 42) and saliva (n = 20) using methylation status of X-linked polymorphic repeats. These genetic data were compared with disease severity based on quantitative clinical parameters. MAIN OUTCOME MEASURES: Visual acuity, Humphrey visual field (HVF) results, full-field electroretinography results, and dark adaptation. RESULTS: Most individuals at all ages were mildly affected or unaffected, whereas those who progressed to moderate or severe vision loss were older than 30 years. RPGR genotype was not associated with clinical severity. The D1264N variant in RPGRIP1L was associated with more severe disease. Skewed XCI toward inactivation of the normal RPGR allele was associated with more severe disease. The XCI ratio in both blood and saliva was a predictor of visual function as measured by HVF diameter, rod amplitude, flicker amplitude, and flicker implicit time. For carriers with extreme XCI skewing of 80:20 or more, 57% were affected severely compared with 8% for those with XCI of less than 80:20 (P = 0.002). CONCLUSIONS: Female carriers with mutations in RPGR demonstrate widely variable clinical severity. X-chromosome inactivation ratios correlate with clinical severity and may serve as a predictor of clinically significant disease. Because RPGR gene therapy trials are underway, a future imperative exists to determine which carriers require intervention and when to intervene. X-chromosome inactivation analysis may be useful for identifying candidates for early intervention.

T Helper 1 Cellular Immunity Toward Recoverin Is Enhanced in Patients With Active Autoimmune Retinopathy.

Autoimmune retinopathy (AIR) causes rapidly progressive vision loss that is treatable but often is confused with other forms of retinal degeneration including retinitis pigmentosa (RP). Measurement of anti-retinal antibodies (ARA) by Western blot is a commonly used laboratory assay that supports the diagnosis yet does not reflect current disease activity. To search for better diagnostic indicators, this study was designed to compare immune biomarkers and responses toward the retinal protein, recoverin, between newly diagnosed AIR patients, slow progressing RP patients and healthy controls. All individuals had measurable anti-recoverin IgG and IgM antibodies by ELISA regardless of disease status or Western blot results. Many AIR patients had elevated anti-recoverin IgG1 levels and a strong cellular response toward recoverin dominated by IFNγ. RP patients and controls responded to recoverin with a lower IFNγ response that was balanced by IL-10 production. Both AIR and RP patients displayed lower levels of total peripheral blood mononuclear cells that were due to reductions of CD4+ TH cells. A comparison of messenger RNA (mRNA) for immune-related genes in whole blood of AIR patients versus RP patients or controls indicated lower expression of ATG5 and PTPN22 and higher expression of several genes involved in TH cell signaling/transcription and adhesion. These data indicate that an immune response toward recoverin is normal in humans, but that in AIR patients the balance shifts dramatically toward higher IFNγ production and cellular activation.

Phenotypic conservation in patients with X-linked retinitis pigmentosa caused by RPGR mutations.

IMPORTANCE: For patients with X-linked retinitis pigmentosa and clinicians alike, phenotypic variability can be challenging because it complicates counseling regarding patients' likely visual prognosis. OBJECTIVE: To evaluate the clinical findings from patients with X-linked retinitis pigmentosa with 13 distinct RPGR mutations and assess for phenotypic concordance or variability. DESIGN: Retrospective medical record review of data collected from 1985 to 2011. SETTING: Kellogg Eye Center, University of Michigan. PATIENTS: A total of 42 patients with X-linked retinitis pigmentosa with mutations in RPGR. Age at first visit ranged from 4 to 53 years, with follow-up ranging from 1 to 11 visits (median follow-up time, 5.5 years; range, 1.4-32.7 years, for 23 patients with >1 visit). MAIN OUTCOMES AND MEASURES: Clinical data assessed for concordance included visual acuity (VA), Goldmann visual fields (GVFs), and full-field electroretinography (ERG). Electroretinography phenotype (cone-rod vs rod-cone dysfunction) was defined by the extent of photopic vs scotopic abnormality. Qualitative GVF phenotype was determined by the GVF pattern, where central or peripheral loss suggested cone or rod dysfunction, respectively. Goldmann visual fields were also quantified and compared among patients. RESULTS: Each mutation was detected in 2 or more related or unrelated patients. Five mutations in 11 patients displayed strong concordance of VA, while 4 mutations in 16 patients revealed moderate concordance of VA. A definitive cone-rod or rod-cone ERG pattern consistent among patients was found in 6 of 13 mutations (46.2%); the remaining mutations were characterized by patients demonstrating both phenotypes or who had limited data or nonrecordable ERG values. Concordant GVF phenotypes (7 rod-cone pattern vs 4 cone-rod pattern) were seen in 11 of 13 mutations (84.6%). All 6 mutations displaying a constant ERG pattern within the mutation group revealed a GVF phenotype consistent with the ERG findings. CONCLUSIONS AND RELEVANCE: While VA and ERG phenotypes are concordant in only some patients carrying identical mutations, assessment of GVF phenotypes revealed stronger phenotypic conservation. Phenotypic concordance is important for establishing proper counseling of patients diagnosed as having X-linked retinitis pigmentosa, as well as for establishing accurate patient selection and efficacy monitoring in therapeutic trials.

Mutations in RPGR and RP2 account for 15% of males with simplex retinal degenerative disease.

PURPOSE: To determine the proportion of male patients presenting simplex retinal degenerative disease (RD: retinitis pigmentosa [RP] or cone/cone-rod dystrophy [COD/CORD]) with mutations in the X-linked retinal degeneration genes RPGR and RP2. METHODS: Simplex males were defined as patients with no known affected family members. Patients were excluded if they had a family history of parental consanguinity. Blood samples from a total of 214 simplex males with a diagnosis of retinal degeneration were collected for genetic analysis. The patients were screened for mutations in RPGR and RP2 by direct sequencing of PCR-amplified genomic DNA. RESULTS: We identified pathogenic mutations in 32 of the 214 patients screened (15%). Of the 29 patients with a diagnosis of COD/CORD, four mutations were identified in the ORF15 mutational hotspot of the RPGR gene. Of the 185 RP patients, three patients had mutations in RP2 and 25 had RPGR mutations (including 12 in the ORF15 region). CONCLUSIONS: This study represents mutation screening of RPGR and RP2 in the largest cohort, to date, of simplex males affected with RP or COD/CORD. Our results demonstrate a substantial contribution of RPGR mutations to retinal degenerations, and in particular, to simplex RP. Based on our findings, we suggest that RPGR should be considered as a first tier gene for screening isolated males with retinal degeneration.

RP2 phenotype and pathogenetic correlations in X-linked retinitis pigmentosa.

OBJECTIVES: To assess the phenotype of patients with X-linked retinitis pigmentosa (XLRP) with RP2 mutations and to correlate the findings with their genotype. METHODS: Six hundred eleven patients with RP were screened for RP2 mutations. From this screen, 18 patients with RP2 mutations were evaluated clinically with standardized electroretinography, Goldmann visual fields, and ocular examinations. In addition, 7 well-documented cases from the literature were used to augment genotype-phenotype correlations. RESULTS: Of 11 boys younger than 12 years, 10 (91%) had macular involvement and 9 (82%) had best-corrected visual acuity worse than 20/50. Two boys from different families (aged 8 and 12 years) displayed a choroideremia-like fundus, and 9 boys (82%) were myopic (mean error, -7.97 diopters [D]). Of 10 patients with electroretinography data, 9 demonstrated severe rod-cone dysfunction. All 3 female carriers had macular atrophy in 1 or both eyes and were myopic (mean, -6.23 D). All 9 nonsense and frameshift and 5 of 7 missense mutations (71%) resulted in severe clinical presentations. CONCLUSIONS: Screening of the RP2 gene should be prioritized in patients younger than 16 years characterized by X-linked inheritance, decreased best-corrected visual acuity (eg, >20/40), high myopia, and early-onset macular atrophy. Patients exhibiting a choroideremia-like fundus without choroideremia gene mutations should also be screened for RP2 mutations. CLINICAL RELEVANCE: An identifiable phenotype for RP2-XLRP aids in clinical diagnosis and targeted genetic screening.

Genetic variants near TIMP3 and high-density lipoprotein-associated loci influence susceptibility to age-related macular degeneration.

We executed a genome-wide association scan for age-related macular degeneration (AMD) in 2,157 cases and 1,150 controls. Our results validate AMD susceptibility loci near CFH (P < 10(-75)), ARMS2 (P < 10(-59)), C2/CFB (P < 10(-20)), C3 (P < 10(-9)), and CFI (P < 10(-6)). We compared our top findings with the Tufts/Massachusetts General Hospital genome-wide association study of advanced AMD (821 cases, 1,709 controls) and genotyped 30 promising markers in additional individuals (up to 7,749 cases and 4,625 controls). With these data, we identified a susceptibility locus near TIMP3 (overall P = 1.1 x 10(-11)), a metalloproteinase involved in degradation of the extracellular matrix and previously implicated in early-onset maculopathy. In addition, our data revealed strong association signals with alleles at two loci (LIPC, P = 1.3 x 10(-7); CETP, P = 7.4 x 10(-7)) that were previously associated with high-density lipoprotein cholesterol (HDL-c) levels in blood. Consistent with the hypothesis that HDL metabolism is associated with AMD pathogenesis, we also observed association with AMD of HDL-c-associated alleles near LPL (P = 3.0 x 10(-3)) and ABCA1 (P = 5.6 x 10(-4)). Multilocus analysis including all susceptibility loci showed that 329 of 331 individuals (99%) with the highest-risk genotypes were cases, and 85% of these had advanced AMD. Our studies extend the catalog of AMD associated loci, help identify individuals at high risk of disease, and provide clues about underlying cellular pathways that should eventually lead to new therapies.

Molecular testing for hereditary retinal disease as part of clinical care.

OBJECTIVE: To describe clinical molecular testing for hereditary retinal degenerations, highlighting results, interpretation, and patient education. METHODS: Mutation analysis of 8 retinal genes was performed by dideoxy sequencing. Pretest and posttest genetic counseling was offered to patients. The laboratory report listed results and provided individualized interpretation. RESULTS: A total of 350 tests were performed. The molecular basis of disease was determined in 133 of 266 diagnostic tests; the disease-causing mutations were not identified in the remaining 133 diagnostic tests. Predictive and carrier tests were requested for 9 and 75 nonsymptomatic patients with known familial mutations, respectively. CONCLUSIONS: Molecular testing can confirm a clinical diagnosis, identify carrier status, and confirm or rule out the presence of a familial mutation in nonsymptomatic at-risk relatives. Because causative mutations cannot be identified in all patients with retinal diseases, it is essential that patients are counseled before testing regarding the benefits and limitations of this emerging diagnostic tool. CLINICAL RELEVANCE: The molecular definition of the genetic basis of disease provides a unique adjunct to the clinical care of patients with hereditary retinal degenerations.

Late-onset macular degeneration and long anterior lens zonules result from a CTRP5 gene mutation.

PURPOSE: To identify the gene responsible for a complex ocular phenotype of late-onset macular degeneration, long anterior zonules (LAZ), and elevated intraocular pressure (IOP) and to study its expression. METHODS: Ocular examination, visual field, fluorescein angiography, and electrophysiology testing were performed. One affected individual was treated with vitamin A. DNA from 55 family members (UM:H389) was used for linkage, mapping, and mutation analysis. Linkage analysis of macular degeneration and LAZ phenotypes was performed independently. Mutations in candidate genes were screened by sequencing. mRNA expression of CTRP5 and MFRP, which are bicistronic genes, was studied by semiquantitative RT-PCR (qRT-PCR) in various human tissues. CTRP5 expression was also evaluated by in situ hybridization. RESULTS: Affected members had LAZ detectable by the third decade and/or macular degeneration by the fourth to fifth decade. A six-month treatment with vitamin A shortened dark adaptation considerably in one affected member. Both conditions mapped independently with zero recombination to 11q23, with maximum lod scores of 3.31 for macular degeneration and 5.41 for LAZ. The same CTRP5 missense mutation was identified in all affected individuals. Retinal pigment epithelium (RPE) and ciliary epithelium (CE) showed highest CTRP5 transcript expression, which was also true for MFRP. CTRP5 tissue expression was confirmed by in situ hybridization. CONCLUSIONS: A single locus at 11q23 is implicated in a complex ocular phenotype involving RPE and CE, tissues of neuroectodermal origin. All individuals with either LAZ and/or macular degeneration carry the same CTRP5 S163R mutation, which is transmitted in autosomal dominant manner.

Late-onset autosomal dominant macular dystrophy with choroidal neovascularization and nonexudative maculopathy associated with mutation in the RDS gene.

PURPOSE: To examine the molecular genetic basis and phenotypic characteristics of an unusual late-onset autosomal dominant macular dystrophy with features of age-related macular degeneration (AMD) in a large family (SUNY901), by using linkage and mutation analyses. METHODS: Blood samples were collected from 17 affected members, 17 clinically unaffected members, and 5 unrelated spouses. Clinical analyses included a review of medical history and standard ophthalmic examination with fundus photography, fluorescein angiography, and electroretinography. Linkage and haplotype analyses were performed with microsatellite markers. Mutation analysis was performed by amplification of exons followed by sequencing. RESULTS: A wide spectrum of clinical phenotypes including exudative and nonexudative maculopathy was observed, with onset in the late fifth decade. Linkage analysis excluded most of the previously known maculopathy loci. Markers D6S1604 (Z(max) of 3.18 at theta = 0), and D6S282 (Z(max) of 3.18 at theta = 0) gave significant positive LOD scores and haplotype analysis localized the disease gene to a 9-centimorgan (cM) interval between markers D6S1616 and D6S459. Mutation analysis excluded the GUCA1A and GUCA1B genes and revealed a missense mutation in the RDS/peripherin gene leading to a Tyr141Cys substitution. A phenotype and haplotype comparison between this and a separate family with the Tyr141Cys mutation suggested the presence of a common ancestral haplotype. CONCLUSIONS: The RDS mutation in codon 141 is associated with an unusual AMD-like late-onset maculopathy. An apparent selective bias was noted favoring the transmission of the mutant allele. These observations broaden the spectrum of phenotypes associated with RDS gene mutations.