Wenhan Yu

Nrl knockdown by AAV-delivered CRISPR/Cas9 prevents retinal degeneration in mice

In retinitis pigmentosa, loss of cone photoreceptors leads to blindness, and preservation of cone function is a major therapeutic goal. However, cone loss is thought to occur as a secondary event resulting from degeneration of rod photoreceptors. Here we report a genome editing approach in which adeno-associated virus (AAV)-mediated CRISPR/Cas9 delivery to postmitotic photoreceptors is used to target the Nrl gene, encoding for Neural retina-specific leucine zipper protein, a rod fate determinant during photoreceptor development. Following Nrl disruption, rods gain partial features of cones and present with improved survival in the presence of mutations in rod-specific genes, consequently preventing secondary cone degeneration. In three different mouse models of retinal degeneration, the treatment substantially improves rod survival and preserves cone function. Our data suggest that CRISPR/Cas9-mediated NRL disruption in rods may be a promising treatment option for patients with retinitis pigmentosa.

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.

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.

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.

A Case Hypersensitive to Bimatoprost and Dexamethasone

PURPOSE The purpose of this study was to report a case hypersensitive to topical bimatoprost and dexamethasone, but with no responsiveness to both latanoprost and travoprost. CASE A 41-year-old Chinese female presented with unilateral glaucoma secondary to iridocyclitis and long-term use of topical steroid. Trabeculectomy only worked for 9 months and then additional topical glaucoma medications were required to control the intraocular pressure (IOP). All commonly used IOP-lowering medications failed, except for bimatoprost, which significantly lowered the IOP. Topical dexamethasone increased IOP and caused ocular hypertension. Ultrasound biomicroscopy (UBM) was used to evaluate the anterior segment of the affected eye. Genomic DNA was extracted for sequence analysis of gene of prostaglandin F receptor (FP), E receptor 1 (EP1) and 2 (EP2) and myocilin. RESULTS UBM revealed cyclodialysis in the patients affected eye after a single dosage of bimatoprost. The cyclodialysis resolved when IOP was elevated with the topical use of dexamethasone. The dexamethasone-induced high IOP could only be controlled by bimatoprost, whereas the bimatoprost-induced low IOP could only be elevated by topical dexamethasone. Allele C of rs3753380 and allele A of rs3766355 in FP gene and a -224T>C variation of myocilin gene were found in this patient. In addition, a novel heterozygous Cys346Tyr mutation was identified in EP2 gene. No sequence variation was found in EP1 gene. CONCLUSIONS The hypersensitivity of the affected eye to topical bimatoprost may be a result, at least in part, of cyclodialysis. The sequence analysis results suggested that, besides the polymorphism of FP gene, there might be some other mechanisms underlying the irresponsiveness of this patient to both latanoprost and travoprost. The mechanisms underlying the bimatoprost-induced cyclodialysis might correlate with its receptor selectivity. The -224T>C variation in the myocilin gene may affect the regulation of expression of this gene by dexamethasone.

Novel compound heterozygous mutations identified in ADAMTSL4 gene in a Chinese family with isolated ectopia lentis

Editor, E ctopia lentis often occurring in association with Marfan’s syndrome, Weill–Marchesani’s syndrome or homocystinuria (Chandra et al. 2014), has been named ‘isolated nonsyndromic ectopia lentis’ (IEL), if it did not occur in association with any apparent systemic disease. Eyes with IEL usually show additional other ocular disorders including cataract, retinal detachment and myopia (Bjerrum & Kessing 1991). IEL was reported to be inherited in an either autosomal dominant pattern (OMIM 129600) or autosomal recessive manner (OMIM 225100) (Ahram et al. 2009). Mutations in the fibrillin 1 (FBN1) gene (OMIM 134797) and the ADAMTSL4 gene (OMIM 610113) were usually responsible (Ades et al. 2004; Ahram et al. 2009). Recently, truncating mutations in the LTBP2 gene were reported as an additional cause of autosomal recessive ectopia lentis as a primary or secondary characteristics in patients with other ocular manifestations (e.g. primary congenital glaucoma) and extraocular manifestations (e.g. marfanoid stature) (Azmanov et al. 2011). Some of the patients with these mutations originated from a Roma/Gypsy founder population. Disease-causing mutation in the ADAMTSL4 gene was first identified by Ahram et al. (2009). Here, we report on a Chinese family in which novel compound heterozygous c.1783dupT and c.2594 G>A mutations of the ADAMTSL4 gene were identified in all affected members. The study included a Chinese family with IEL with autosomal recessive inheritance. The study was approved by the Medical Ethics Committee of the West China Hospital of Sichuan University. Informed consent was obtained from all participants. Family recruitment and clinical examination included nine members of two generations in this family. Four family members of seven of the second generation were affected. Poor vision since childhood was noticed in all affected individuals. Proband II:1, a 21-yearold man (Fig. 1A,B) presented with bilateral lens subluxation into the temporal direction and lens opacity. The left eye additionally showed signs of a secondary-lens-induced pupillary block glaucoma with an intraocular pressure of 51 mmHg and secondary iris atrophy. The axial length of the

266. In Vivo Rod Photoreceptor Reprogramming Using AAV-Delivered CRISPR/Cas9 Rescues Retinal Degeneration

Retinitis pigmentosa (RP) is the most common form of inherited retinal dystrophy and the leading cause of inherited blindness, due to mutations in any of the over 60 genes/loci identified so far. The disease is characterized by an initial loss of rod photoreceptors and secondary cone cell death. Since cone photoreceptors are responsible for day time vision and visual acuity, preserving cone functions in RP patients is a priority when developing treatment strategies. NRL is a transcription factor that determines the rod photoreceptor cell fate during retinal development. Acute gene knockout of Nrl in mice was shown to reprogram adult rods into cone-like cells, rendering them resistant to effects of mutations in rod-specific genes and consequently preventing secondary cone loss (Montana CL, et al. PNAS, 2013; 110: 1732-7). With a goal to develop this approach for treatment of RP, we used adeno-associated virus (AAV)-delivered CRISPR/Cas9 for Nrl-knockdown in rod photoreceptors. AAV vectors were constructed to carry a photoreceptor-specific Cas9 nuclease expression cassette or a single-guided RNA (sgRNA) targeting Nrl or eGFP gene. The Cas9 and the sgRNA vectors were co-delivered into mice by subretinal administration. Potency of the AAV-CRISPR/Cas9 system was validated by EGFP knockdown in a mouse line with eGFP-labeled rods. Nrl knockdown was conducted in wild-type C57/Bl6 or Crxp-Nrl, a mouse line with rod-only photoreceptors. Molecular, histological and functional alterations were examined by next generation sequencing, immunoblot analysis, immunofluorescence, electron microscopy, and electroretinography (ERG). Our results showed that eGFP and Nrl were efficiently knocked down following AAV-CRISPR/Cas9 treatment. For Nrl knockdown, almost all insertions and deletions were detected in the targeted Nrl locus, and very few mutations were identified in ten potential off-target loci. A majority of the transduced rods acquired characteristics of cone photoreceptors following Nrl-CRISPR/Cas9 vector treatment, as demonstrated by reduced expression of rod-specific genes and enhanced expression of cone-specific genes, loss of the unique rod chromatin pattern, and diminished rod ERG response. Rescue of retinal degeneration was assessed in three mouse models harboring either recessive or dominant rod-specific mutations. In all three models, the Nrl-CRISPR/Cas9 vector treated eyes maintained significantly better photoreceptor viability and cone function than control eyes, as revealed by remarkably thicker photoreceptor layer, higher cone cell number, greater cone ERG amplitude and better optomotor behavior. In conclusion, AAV-CRISPR-mediated Nrl gene knockdown can efficiently reprogram rods into cone-like photoreceptors and prevent secondary cone death in retinal degeneration, which could be developed into a viable treatment for RP in humans.

Studies of a pedigree with limbal dermoid cyst.

AIM To study clinical features and gene mutations within the paired-like homeodomain transcription factor 2 (PITX2) gene in a pedigree of bilateral limbal dermoids. METHODS Complete eye examinations have been performed on each individual of the family. Exons of paired-like homeodomain transcription factor 2 (PITX2) were amplified by polymerase chain reaction, sequenced, and compared with a reference database. RESULTS We described the phenotype, clinic findings in a family with two affected members. The masses of the probands eyes were excised surgically demonstrating a dermoid cyst by histopathological examination. No mutation was detected in the gene PITX2 in this pedigree. CONCLUSION A family of limbal dermoid cyst was reported. In addition, no pathogenic sequence variations were found in PITX2, indicating that this phenotype in this family is a distinctive entity.

TGFBI gene mutation analysis in a Chinese pedigree of Avellino corneal dystrophy

AIM To analyze phenotype and genotype of a Chinese pedigree with Avellino corneal dystrophy (ACD). METHODS Complete ophthalmic examinations were performed on all the family members. Exons of TGFBI were amplified by polymerase chain reaction, sequenced, and compared with a reference database. RESULTS A single heterozygous G>A (R124H) point mutation was identified in exon 4 of TGFBI in three affected members and two unaffected children who were offsprings of the affected members, but not in the other family members. CONCLUSION Mutation R124H in TGFBI was identified in this pedigree and appeared to be the disease causing mutation. Atypical phenotype and low penetrance was observed in this pedigree.