Next-Generation Sequencing-Aided Rapid Molecular Diagnosis of Occult Macular Dystrophy in a Chinese Family.

Purpose: To show early, rapid and accurate molecular diagnosis of occult macular dystrophy (OMD) in a four-generation Chinese family with inherited macular dystrophy. Methods: In the current study, we comprehensively screened 130 genes involved in common inherited non-syndromic eye diseases with next-generation sequencing-based target capture sequencing of the proband of a four-generation Chinese family that has suffered from maculopathy without a definitive diagnosis for over 10 years. Variants were filtered and analyzed to identify possible disease-causing variants before validation by Sanger sequencing. Results: Two heterozygous mutations-RP1L1 c.133 C > T (p.Arg45Trp), which is a hot spot for OMD, and ABCA4 c.6119 G > A (p.Arg2040Gln), which was identified in Stargardt's disease were found in three patients, but neither of the mutations was found in the unaffected individuals in the same family, who are phenotypically normal or in the normal control volunteers. Conclusion: These results cannot only confirm the diagnosis of OMD in the proband, but also provide presymptomatic diagnosis of the proband's children before the onset of visual acuity impairment and guidance regarding the prognosis and management of these patients. Heterozygous mutations of RP1L1 c.133 C > T (p.Arg45Trp) and ABCA4 c.6119 G > A (p.Arg2040Gln) are likely responsible for OMD. Our results further extend our current understanding of the genetic basis of OMD, and emphasize the importance of molecular diagnosis and genetic counseling for OMD.

Identification of novel mutations by targeted exome sequencing and the genotype-phenotype assessment of patients with achromatopsia.

BACKGROUND: Achromatopsia (ACHM) is a severe congenital autosomal recessive retinal disorder caused by loss of cone photoreceptors. Here, we aimed to determine the underlying genetic lesions and phenotypic correlations in two Chinese families with ACHM. METHODS: Medical history and clinical evaluation were obtained from both families. Targeted exome sequencing (TES) was performed on 201 disease-causing genes of inherited retinal dystrophies to screen for ACHM causative mutations in the two probands. RESULTS: The compound heterozygous mutations in CNGA3 (c.1074G > A, p.W358X; c.1706G > A, p.R569H) were identified in the first proband, and a novel homozygous mutation (c.968C > A, p.A323D) was detected in the other pedigree. The proposed topological model of the CNGA3 polypeptide suggested that the missense mutations primarily affected the transmembrane helix 5 and the cGMP-binding domain, respectively. Crystal structure modeling of the cyclic nucleotide-gated cation channel α-3 (CNGA3) protein encoded by the CNGA3 gene revealed an abnormal combined structure generated by R569H. CONCLUSIONS: We firstly used the TES approach to identify genetic alterations in patients with ACHM. We uncovered three mutations in CNGA3, including one novel mutation. Our results not only expand the genotypic spectrum for CNGA3 mutations, but also demonstrate that the TES approach is a valuable tool for molecular diagnosis.

R102W mutation in the RS1 gene responsible for retinoschisis and recurrent glaucoma.

AIM: To identify the mutations in RS1 gene associated with typical phenotype of X-linked juvenile retinoschisis (XLRS) and a rare condition of concomitant glaucoma. METHODS: Complete ophthalmic examinations were performed in the proband. The coding regions of the RS1 gene that encode retinoschisin were amplified by polymerase chain reaction and directly sequenced. RESULTS: The proband showed a typical phenotype of XLRS with large peripheral retinal schisis in both eyes, involving the macula and combined with foveal cystic change, reducing visual acuity. A typical phenotype of recurrent glaucoma with high intraocular pressure (IOP) and reduced visual field was also demonstrated with the patient. Mutation analysis of RS1 gene revealed R102W (c.304C>T) mutations in the affected male, and his mother was proved to be a carrier with the causative mutation and another synonymous polymorphism (c.576C>CT). CONCLUSION: We identified the genetic variations of a Chinese family with typical phenotype of XLRS and glaucoma. The severe XLRS phenotypes associated with R102W mutations reveal that the mutation determines a notable alteration in the function of the retinoschisin protein. Identification of the disease-causing mutation is beneficial for future clinical references.

Electrical stimulation ameliorates light-induced photoreceptor degeneration in vitro via suppressing the proinflammatory effect of microglia and enhancing the neurotrophic potential of Müller cells.

Many types of electrical stimulation (ES) devices have been shown to promote the survival of degenerated neural cells, such as dopaminergic neurons in the medial forebrain bundle-transected rats, ischemic-injured cortical neurons and inner-and outer-nuclear-layer cells in degenerated retina. Using a rat photic injury model, our lab previously proved the neuroprotective effect of transcorneal electrical stimulation (TCES) on apoptotic photoreceptor cells. To delineate the mechanisms that might underlie this process, the effects of ES on light-damaged photoreceptor degeneration-induced microglia and Müller cell activation were investigated in the present in vitro study. Our data showed that ES (3 ms, 20 Hz, 300-1600 µA) increased survival among light-reared cone-derived cells (661W) cultured alongside microglia or Müller cells analyzed by LDH and TUNEL assays. The degree of rescue was found to depend on the different intensities of the ES current. The immunocytochemistry revealed that ES significantly decreased the numbers of activated microglia cells with ameboid shapes and increased the numbers of reactive gliotic Müller cells with larger soma when they were co-cultured with light-damaged 661W cells. Real-time RT-PCR and Western blotting indicated that ES which was applied to different co-cultures and 661W cell-conditioned media (661WCM)-treated glia cultures had a prominent inhibitive effect on the secretion of interleukin (IL)-1ß and tumor necrosis factor (TNF)-α in microglia and a positive regulative effect on the production of brain-derived neurotrophic factor (BDNF) and ciliary neurotrophic factor (CNTF) in Müller cells. The death rate of light-exposed 661W cells cultured with microglia was decreased significantly by the addition of neutralizing antibodies against IL-1ß and TNF-α. On the other hand, the death rate of light-exposed 661W cells cultured with Müller cells was prominently increased when the co-culture was incubated in the presence of neutralizing antibody against BDNF while anti-CNTF neutralizing antibody did not induce additional exacerbation of the cell death among those 661W cells. These findings indicate the feasibility of using ES to create a nurturing environment for light-damaged photoreceptor cells. This environment is characterized by diminished microglial activation and fortified Müller cells reactive gliosis, which may have great potential in ameliorating photoreceptor damage. In this way, ES was here determined to be a novel, potent therapeutic option for delaying the progression of photoreceptor degeneration in patients suffering from retinitis pigmentosa (RP).

Novel RPGR-ORF15 mutations in X-linked retinitis pigmentosa patients.

X-linked retinitis pigmentosa (XLRP) is the most severe type of retinitis pigmentosa (RP), with patients consistently showing early onset and rapid deterioration. Obtaining a genetic diagnosis for a family with XLRP is important for counseling purposes. In this study, we aimed to identify disease-causing mutations in two unrelated XLRP families. Genetic analysis was performed on two unrelated XLRP families. Genomic DNA was extracted from peripheral blood or amniotic fluid samples. The coding regions and intron/exon boundaries of the Retinitis Pigmentosa GTPase Regulator (RPGR) and RP2 genes were amplified by PCR and then sequenced directly. A clinically unaffected pregnant female and the four month old fetus were found to have a hemizygous 2 base pair deletion (g.ORF15+484_485delAA) in the exon ORF15 of RPGR gene. In another XLRP family, a nonsense mutation (g.ORF15+810G>T) was identified. Neither mutation has been reported previously. Both are predicted to cause premature termination of the protein. In conclusion, we identified a micro-deletion through prenatal genetic diagnosis and another novel nonsense mutation in RPGR-ORF15. Identifying a disease-causing mutation facilitated early diagnosis and genetic counseling for the patients. Discovery of novel mutations also broadens knowledge of XLRP and the spectrum of its pathogenic genotypes.

Neuroprotective effect of transcorneal electrical stimulation on light-induced photoreceptor degeneration.

Direct electrical stimulation of neural tissues is a strategic approach to treat injured axons by accelerating their outgrowth [Al-Majed, A.A., Neumann, C.M., Brushart, T.M., Gordon, T., 2000. Brief electrical stimulation promotes the speed and accuracy of motor axonal regeneration. J. Neurosci. 20, 2602-2608] and promoting their regeneration [Geremia, N.M., Gordon, T., Brushart, T.M., Al-Majed, A.A., Verge, V.M.K., 2007. Electrical stimulation promotes sensory neuron regeneration and growth-associated gene expression. Exp. Neurol. 205, 347-359]. Recently, transcorneal electrical stimulation (TCES), a novel less invasive method, has been shown to rescue axotomized and damaged retinal ganglion cells [Morimoto, T., Miyoshi, T., Matsuda, S., Tano, Y., Fujikado, T., Fukuda, Y., 2005. Transcorneal electrical stimulation rescues axotomized retinal ganglion cells by activating endogenous retinal IGF-1 system. Invest. Ophthalmol. Vis. Sci. 46(6), 2147-2155]. Here, we investigated the neuroprotection of TCES on light-induced photoreceptor degeneration and the underlying mechanism. Adult male Sprague-Dawley (SD) rats received TCES before (pre-TCES) or after (post-TCES) intense light exposure. After fourteen days of light exposure, retinal histology and electroretinography were performed to evaluate the neuroprotective effect of TCES. The mRNA and protein levels of apoptotic-associated genes including Bcl-2, Bax, Caspase-3 as well as ciliary neurotrophic factor (CNTF) and brain-derived neurotrophic factor (BDNF) in the retinas were determined by real-time PCR and Western blot analysis. The localization of these gene products in the retinas was examined by immunohistochemistry. Both pre- and post-TCES ameliorated the progressive photoreceptor degeneration. The degree of rescue depended on the strength of the electric charge. Post-TCES showed a relatively better and longer-term protective effect than pre-TCES. Real-time PCR and Western blot analysis revealed an upregulation of Bcl-2, CNTF, and BDNF and a downregulation of Bax in the retinas after TCES. Immunohistochemical studies showed that Bcl-2 and CNTF were selectively upregulated in Müller cells. These findings provide a new therapeutic method to prevent or delay photoreceptor degeneration through activating the intrinsic survival system.

Macular hole formation in patients with retinitis pigmentosa and prognosis of pars plana vitrectomy.

PURPOSE: To investigate macular hole (MH) formation in patients with retinitis pigmentosa (RP) and to determine surgical prognosis for these patients. METHODS: Data for four RP patients with MHs were retrospectively reviewed. Comprehensive ocular examinations, including electroretinography, Goldmann perimetry, color funduscopy, B-scan ultrasonic tomography, and optical coherence tomography (OCT), were performed. All patients underwent par plana vitrectomy followed by peeling of the posterior inner limiting membrane and gas tamponade. Preoperative best-corrected visual acuity ranged from 20/1000 to 20/100. RESULTS: Typical full-thickness MHs were evident by OCT for three patients. Two of these patients had posterior vitreous detachment or vitreoretinal traction; one had cystoid macular edema in the fellow eye. A fourth patient had extensive retinal detachment; ophthalmoscopy revealed a hole in the macular region. After surgery, significant improvement of visual acuity was recorded for three patients; the patient with retinal detachment had no change in visual acuity in the affected eye. OCT showed adequate sealing of the MHs. CONCLUSIONS: We report results of a clinical investigation including four RP patients with concomitant MHs. Vitreoretinal surgery is necessary for effective management of MHs in RP.

[Analysis of ophthalmic projects granted by National Natural Science Foundation].

OBJECTIVE: To understand the status of basic research work in the field of ophthalmology by analyzing the projects funded by the National Natural Science Foundation of China (NSFC) from the year of 1986 to 2007, and offer as a reference to the ophthalmologists and researchers. METHODS: NSFC supported ophthalmology projects in the 22 year's period were collected from the database of NSFC. The field of funded projects, the research team and their achievements were analyzed. RESULTS: There were 228 applicants from 47 home institutions were funded in the field of ophthalmology during the past 22 years, 323 projects funded with 66.74 million Yuan in total, in which 165 projects were fulfilled before the end of 2006. The applied and funded projects mainly focus on six different kinds of research area related to retinal diseases, corneal diseases, glaucoma, optic nerve diseases, myopia and cataract, and 70% of them were basic research in nature. As a brief achievement of 165 fulfilled projects, more than 610 papers were published in domestic journals, over 140 papers were published in Science Citation Index journals, more than 600 people were trained, and over 20 scientific awards were obtained. CONCLUSION: The number of funded projects and achievement of fulfilled projects in the discipline of ophthalmology gradually increased over the past two decades, the research fields were concentrated in certain diseases. NSFC has played an important role in promoting the development of ophthalmology research and bringing up specialists in China. However, clinical research, continuously research, transforming from basic research to clinic applications and multidisciplinary cross studies should be strengthened.