Suping Cai

Exome Sequencing Identifies Compound Heterozygous Mutations in CYP4V2 in a Pedigree with Retinitis Pigmentosa

Retinitis pigmentosa (RP) is a heterogeneous group of progressive retinal degenerations characterized by pigmentation and atrophy in the mid-periphery of the retina. Twenty two subjects from a four-generation Chinese family with RP and thin cornea, congenital cataract and high myopia is reported in this study. All family members underwent complete ophthalmologic examinations. Patients of the family presented with bone spicule-shaped pigment deposits in retina, retinal vascular attenuation, retinal and choroidal dystrophy, as well as punctate opacity of the lens, reduced cornea thickness and high myopia. Peripheral venous blood was obtained from all patients and their family members for genetic analysis. After mutation analysis in a few known RP candidate genes, exome sequencing was used to analyze the exomes of 3 patients III2, III4, III6 and the unaffected mother II2. A total of 34,693 variations shared by 3 patients were subjected to several filtering steps against existing variation databases. Identified variations were verified in the rest family members by PCR and Sanger sequencing. Compound heterozygous c.802-8_810del17insGC and c.1091-2A>G mutations of the CYP4V2 gene, known as genetic defects for Bietti crystalline corneoretinal dystrophy, were identified as causative mutations for RP of this family.

H-1152 Effects on Intraocular Pressure and Trabecular Meshwork Morphology of Rat Eyes

PURPOSE The aim of this study was to elucidate the effects of the Rho-kinase inhibitor, H-1152, on cultured human trabecular meshwork (HTM) cells, TM morphology, and intraocular pressure (IOP) in rats. METHODS Cultured HTM cells were treated with H-1152. Changes in cell morphology and the organization of the actin cytoskeleton and focal adhesions were evaluated by microscopy and immunofluorescence. H-1152 was administered topically to the eyes of conscious rats, and IOP was measured with a commercially available tonometer before and after treatment. The eyes were enucleated 1 h after treatment, fixed, and processed for morphologic analysis by light and electron microscopy. RESULTS Exposure of the cultured HTM cells to 20 microM of H-1152 induced elongation and separation of cells, deterioration, and loss of actin stress fibers and focal adhesions within 2 h. Topical administration of H-1152 resulted in a significant decrease in IOP from 0.5 to 6 h, with the maximum IOP reduction of 28.1% at 1 h post-treatment (P < 0.001; n = 10). H-1152 caused an expansion of the intercellular spaces and loss of extracellular material in the juxtacanalicular region of the TM in rat eyes. CONCLUSIONS The IOP-lowering effect of H-1152 in rat eyes is likely due to changes in TM-cell morphology, the actin cytoskeleton, and cellular adhesions in the conventional outflow pathway. H-1152 has potential as a new antiglaucoma medication.

Neuroprotective effects of C-type natriuretic peptide on rat retinal ganglion cells.

PURPOSE. To evaluate the potential neuroprotective effects of C-type natriuretic peptide (CNP) on rat retinal ganglion cells (RGCs). METHODS. Cultured adult rat retinal cells were treated with vehicle, CNP, or atrial natriuretic peptide (ANP), followed by cytotoxic insults (glutamate, TNFalpha, or withdrawal of trophic factor). RGC survival was analyzed by counting Thy-1-positive cells in each well. For in vivo evaluation, N-methyl-d-aspartate (NMDA) with or without CNP was injected intravitreally into rat eyes. At various time points after injection, retinal cross-sections were analyzed for thickness changes in the retinal layers, and retinal flat mounts were assessed by counting cresyl violet-labeled or TUNEL-positive cells. Expressions of natriuretic peptide receptor-B (NPRB) and apoptosis-related genes in retina, including Bcl-xL, BAX, and micro-calpain, were analyzed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). RESULTS. At 50 and 500 nM, CNP, but not ANP, significantly (P < 0.05) protected against glutamate-insult and trophic factor withdrawal-induced RGC death in vitro. Neither peptide significantly affected TNFalpha-induced cytotoxicity. Intravitreal injection of NMDA (20 nanomoles) significantly (P < 0.05) decreased the thickness of the inner plexiform layer (IPL), induced cell loss, increased the number of TUNEL-positive cells in the RGC layer, and upregulated the expression of Bcl-xL, BAX, and micro-calpain. All these effects were significantly (P < 0.05) alleviated by concomitant injection of CNP (4.5 nmol, 10 microg). The neuroprotective effects of CNP were maintained up to 14 days after CNP injection. CONCLUSIONS. CNP protects rat RGCs against the apoptotic damage induced by insults such as excitatory amino acid, both in vitro and in vivo.

Is low dose of Estrogen beneficial for prevention of glaucoma

Glaucoma, as characterized by accelerated retinal ganglion cell (RGC) death and cupping of optic nerve head (ONH), is one of the leading causes of blindness worldwide. Elevated intraocular pressure (IOP) is generally considered as a major risk factor in the pathogenesis of glaucoma. Previous studies showed that glaucoma caused decrease in collagen and elastin density in several ocular tissues, such as lamina cribrosa, peripapillary sclera and cornea, and resulted in reduced elasticity and compliance of these tissues. It is known that estrogen has protective effects against glaucoma, yet the underlying mechanism still remains obscure. Prior researches have provided evidences showing that the estrogen receptors (ERs) express in a variety of the ocular tissues. Estrogen activates the synthesis of collagen fiber and improves the compliance of these tissues. This leads to a reasonable postulation that increased estrogen may result in a higher content of the collagen fibers and enhanced flexibility of the whole eye, which would therefore decrease IOP. Particularly, the increase in the amounts of collagen fibers at lamina cribrosa improves its compliance, which in turn relieves its compression on RGC axons. Therefore, even at the same IOP level, the softening of cribriform foramina yields a more flexible environment for the RGCs to survive. We therefore hypothesize that estrogen at proper dosage can be considered as a potential therapy for glaucoma since it is able to prevent the eye from glaucomatous damage and lower IOP, especially for those menopausal women with glaucoma.

Myopia: A collagen disease?

Myopia is an increasingly important public health problem in the world. Even though previous studies have strongly implicated a role of certain environmental factors such as visual near-work in myopia development, the pathogenesis of this disease still remains unclear. There is evidence showing that myopia is primarily a hereditary condition, combined with or without environmental influence or individual habitual factors. Recent research suggests that collagens in the sclera play an important role in the development of myopia. Based on a literature review after a Medline search on articles on myopia, changes in scleral collagen appeared to underlie or be associated with the pathogenetic factors (including inheritance) involved in myopia development. Therefore, we hypothesized that myopia is a disorder, in which alterations of scleral collagens may be responsible for the pathological changes found in it.

Single nucleotide polymorphism of MYOC affected the severity of primary open angle glaucoma

AIM To detect the mutations in two candidate genes, myocilin (MYOC) and cytochrome P450 1B1 (CYP1B1), in a Chinese family with primary open angle glaucoma (POAG). METHODS The family was composed of three members, the parents and a daughter. All members of the family underwent complete ophthalmologic examinations. Exons of MYOC and CYP1B1 genes were screened for sequence alterations by polymerase chain reaction (PCR) and direct DNA sequencing. RESULTS The mother was the proband, she was diagnosed as POAG in both eyes. Her daughter was diagnosed as juvenile-onset POAG. The father was asymptomatic. One MYOC heterozygous mutation c.1150 G>A (D384N) in exon 3 was identified in the mother, another MYOC heterozygous variation c.1058 C>T (T353I) in exon 3 was identified in the father, and the daughter inherited both of the variations. Meanwhile, three single nucleotide polymorphisms (SNPs) in CYP1B1 gene were found in the family. CONCLUSION The D384N mutation of MYOC has been reported as one of disease-causing mutations in POAG, whereas T353I variation of MYOC was thought as a high risk factor for POAG. The two variations of MYOC were first reported in one juvenile-onset POAG patient who presented with more severe clinical manifestations, suggesting that T353I polymorphism of MYOC may be associated with the severity of POAG.

A Novel Nonsense Mutation of the GPR143 Gene Identified in a Chinese Pedigree with Ocular Albinism

Background The purpose of this study was to elucidate the molecular basis of ocular albinism type I in a Chinese pedigree. Methodology/Principal Findings Complete ophthalmologic examinations were performed on 4 patients, 7 carriers and 17 unaffected individuals in this five-generation family. All coding exons of four-point-one (4.1), ezrin, radixin, moesin (FERM) domain-containing 7 (FRMD7) and G protein-coupled receptor 143 (GPR143) genes were amplified by polymerase chain reaction (PCR), sequenced and compared with a reference database. Ocular albinism and nystagmus were found in all patients of this family. Macular hypoplasia was present in the patients including the proband. A novel nonsense hemizygous mutation c.807T>A in the GPR143 gene was identified in four patients and the heterozygous mutation was found in seven asymptomatic individuals. This mutation is a substitution of tyrosine for adenine which leads to a premature stop codon at position 269 (p.Y269X) of GPR143. Conclusions/Significance This is the first report that p.Y269X mutation of GPR143 gene is responsible for the pathogenesis of familial ocular albinism. These results expand the mutation spectrum of GPR143, and demonstrate the clinical characteristics of ocular albinism type I in Chinese population.

Transformation of human trabecular meshwork cells with SV40 TAg alters promoter utilization

Purpose. To compare promoter usage in primary differentiated and SV40 TAg transformed human trabecular meshwork cells (HTM and TM1 cells). Methods. Cultured HTM and TM1 cells were transfected with vectors expressing MYOC/TIGR from the CMV-IE, IE4/5 (HSV immediate early 4/5), ICP6 (early gene ICP6 of HSV), EF-1a (human elongation factor 1a-subunit), or the UB6 (human ubiquitin) promoters, respectively. Immunoblotting was used to measure MYOC/TIGR protein expression. MYOC/TIGR expression at the RNA level was detected by Northern blotting. Results. In primary HTM cells, CMV was the only promoter displaying substantial activity. In TM1 cells, several promoters were functional with the order in decreasing activity being EF-1a = CMV = UB6 >> IE4/5. Conclusions. The difference between the normal and transformed HTM cells suggests that the latter cell type has alterations that influence cellular promoter function. The type of cell used is likely to be a crucial factor in evaluating the functions of promoter elements for genes expressed in the trabecular meshwork and in screening promoters for use in gene delivery studies, especially for evaluations of the MYOC/TIGR gene in relation to glaucoma mechanisms.

Experimental Tibetan monkey domestication and its application for intraocular pressure measurement.

AIM To train Tibetan monkey (Macaca thibetana) for intraocular pressure (IOP) measurement in conscious state and obtain normal IOP in conscious Tibetan Macaque. METHODS The training was based on award-conditioned behavior. Food stimulation and human-animal interaction were used in this training. RESULTS Trained Tibetan monkeys calmly accepted IOP measurement by the TonoVet® rebound tonometer without sedation or anesthesia and their IOP values were similar to other primates. CONCLUSION Human-cultivated Thibetan monkeys are tamable, and can be used for biomedical research such as ophthalmic research without anesthesia.

Possible mechanism for the gastro-intestinal adverse effects upon topical application of Prostaglandin F2α analogs

Prostaglandin F(2)α analogs (PGAs), including latanoprost, travoprost and bimatoprost, the first choice for the pharmaceutical treatment of glaucoma, are gaining more attention on their systemic side effects in recent years. The gastro-intestinal effects are among the most reported adverse effects upon topical application of PGAs. Yet, the underlying mechanism remains to be unknown. In the current study, we performed a molecular genetic analysis on the patient reported by Yu et al. (BMJ Case Rep, 2009), who developed nausea, vomiting and diarrhea after topical application of travoprost and latanoprost, but not bimatoprost, and then speculated that the mechanism underlying the gastro-intestinal distress secondary to PGA topical application should be attributed to their stimulation of smooth muscles of the gastric and intestinal tract via prostanoid receptors. We postulate that the diversified receptor selectivity of various PGAs might mediate their diversified gastro-intestinal effects. To further verificate the speculation, other three glaucoma patients who exhibited different gastro-intestinal responses to different PGA medications were enrolled. The results suggested that the relative expression level of FP receptor, versus EP receptors, might be associated with the severity of gastro-intestinal effects incurred by PGAs. Owing to the differed expression levels of FP receptor, the responses of various patients to different PGAs can be variable.