Lactylation-driven FTO targets CDK2 to aggravate microvascular anomalies in diabetic retinopathy.

Diabetic retinopathy (DR) is a leading cause of irreversible vision loss in working-age populations. Fat mass and obesity-associated protein (FTO) is an N6-methyladenosine (m6A) demethylase that demethylates RNAs involved in energy homeostasis, though its influence on DR is not well studied. Herein, we detected elevated FTO expression in vitreous fibrovascular membranes of patients with proliferative DR. FTO promoted cell cycle progression and tip cell formation of endothelial cells (ECs) to facilitate angiogenesis in vitro, in mice, and in zebrafish. FTO also regulated EC-pericyte crosstalk to trigger diabetic microvascular leakage, and mediated EC-microglia interactions to induce retinal inflammation and neurodegeneration in vivo and in vitro. Mechanistically, FTO affected EC features via modulating CDK2 mRNA stability in an m6A-YTHDF2-dependent manner. FTO up-regulation under diabetic conditions was driven by lactate-mediated histone lactylation. FB23-2, an inhibitor to FTO's m6A demethylase activity, suppressed angiogenic phenotypes in vitro. To allow for systemic administration, we developed a nanoplatform encapsulating FB23-2 and confirmed its targeting and therapeutic efficiency in mice. Collectively, our study demonstrates that FTO is important for EC function and retinal homeostasis in DR, and warrants further investigation as a therapeutic target for DR patients.

ALKBH5 causes retinal pigment epithelium anomalies and choroidal neovascularization in age-related macular degeneration via the AKT/mTOR pathway.

Retinal pigment epithelium (RPE) dysfunction and choroidal neovascularization (CNV) are predominant features of age-related macular degeneration (AMD), with an unclear mechanism. Herein, we show that RNA demethylase α-ketoglutarate-dependent dioxygenase alkB homolog 5 (ALKBH5) is up-regulated in AMD. In RPE cells, ALKBH5 overexpression associates with depolarization, oxidative stress, disturbed autophagy, irregular lipid homeostasis, and elevated VEGF-A secretion, which subsequently promotes proliferation, migration, and tube formation of vascular endothelial cells. Consistently, ALKBH5 overexpression in mice RPE correlates with various pathological phenotypes, including visual impairments, RPE anomalies, choroidal neovascularization (CNV), and interrupted retinal homeostasis. Mechanistically, ALKBH5 regulates retinal features through its demethylation activity. It targets PIK3C2B and regulates the AKT/mTOR signaling pathway with YTHDF2 as the N6-methyladenosine reader. IOX1, an ALKBH5 inhibitor, suppresses hypoxia-induced RPE dysfunction and CNV progression. Collectively, we demonstrate that ALKBH5 induces RPE dysfunction and CNV progression in AMD via PIK3C2B-mediated activation of the AKT/mTOR pathway. Pharmacological inhibitors of ALKBH5, like IOX1, are promising therapeutic options for AMD.

JP1, a polypeptide specifically targeting integrin αVß3, ameliorates choroidal neovascularization and diabetic retinopathy in mice.

Anti-vascular endothelial growth factor (VEGF) drugs have revolutionized the treatment of neovascular eye diseases, but responses are incomplete in some patients. Recent evidence shows that integrins are involved in the pathogenesis of neovascular age-related macular degeneration and diabetic retinopathy. JP1, derived from an optimized seven-amino-acid fragment of JWA protein, is a polypeptide specifically targeting integrin αVß3. In this study we evaluated the efficacy of JP1 on laser-induced choroidal neovascularization (CNV) and retinal vascular leakage. CNV mice received a single intravitreal (IVT) injection of JP1 (10, 20, 40 µg) or ranibizumab (RBZ, 10 µg). We showed that JP1 injection dose-dependently inhibited laser-induced CNV; the effect of RBZ was comparable to that of 20 µg JP1; a combined IVT injection of JP1 (20 µg) and RBZ (5 µg) exerted a synergistic effect on CNV. In the 3rd month after streptozotocin injection, diabetic mice receiving IVT injection of JP1 (40 µg) or RBZ (10 µg) once a week for 4 weeks showed significantly suppressed retinal vascular leakage. In both in vivo and in vitro experiments, JP1 counteracted oxidative stress and inflammation via inhibiting ROS/NF-κB signaling in microglial cells, and angiogenesis via modulating MEK1/2-SP1-integrin αVß3 and TRIM25-SP1-MMP2 axes in vascular endothelial cells. In addition, intraperitoneal injection of JP1 (1, 5 or 10 mg) once every other day for 3 times also dose-dependently inhibited CNV. After intraperitoneal injection of FITC-labeled JP1 (FITC-JP1) or FITC in laser-induced CNV mice, the fluorescence intensity in the CNV lesion was markedly increased in FITC-JP1 group, compared with that in FITC group, confirming that JP1 could penetrate the blood-retinal barrier to target CNV lesion. We conclude that JP1 can be used to design novel CNV-targeting therapeutic agents that may replace current invasive intraocular injections.

Chordin-like 2 influences the differentiation fate of retinal pigment epithelium cells by dynamically regulating BMP pathway.

AIM: To explore the functions of Chordin-like 2, which is encoded by CHRDL2, in the process of retinal pigmented epithelium (RPE) differentiation and damage repair. METHODS: The fetal RPE cells (fRPE) was obtained from aborted fetus which obeyed medical ethics. Real-time quantitative polymerase chain reaction was used to measure expression quantity of CHRDL2 and other functional genes expression. Knocking down and overexpression was used to analyze the functions about Chordin-like 2. Enzyme-linked immunosorbent assay (ELISA) was used to detect the secretion of bone morphogenetic proteins 4 (BMP4). Flow cytometry was used to analyze cell cycle. Cell morphology was observed by phase contrast microscope (PCM). RESULTS: In normal RPE cells, CHRDL2 was firstly upregulated and followed a downregulation but eventually, it was expressed higher than the cells which undergone epithelial-mesenchymal transition (EMT). After knocking down CHRDL2, the secretion of BMP4 was decreased, RPE-related genes (OTX2, MITF, RPE65) were downregulated while EMT-related genes (SNAI1, VIM) were upregulated. However, the expression of these related genes after overexpression of CHRDL2 had contrary results. Chordin-like 2 also regulated the cell cycle by regulating BMP pathway. When CHRDL2 was knocked down, more fRPE cells stayed in S phase of cell cycle, while adding BMP4 reduced the proportion of the cells in S phase. However, overexpression of CHRDL2 increased more BMP4 secretion, this effect decreased the number of cells in S phase, but exogenous BMP inhibitor also could change this effect. At last, in the process of RPE cells differentiation, adding BMP4 at early stage could intervene normal RPE differentiation. Compared with BMP4, inhibiting BMP pathway had no significant negative effect at early stage, but suppressed differentiation at late stage. CONCLUSION: BMP pathway can be activated in a correct temporal order, otherwise, the cells have incorrect differentiation orientation. And Chordin-like 2 plays a role in dynamic regulation of BMP pathway and it also regulates the differentiation of RPE cells. Therefore, this research enlightens a new direction to inhibit EMT and promote cell redifferentiation after injury.

Displacement of the retina after idiopathic macular hole surgery with different internal limiting membrane peeling patterns.

AIM: To explore retinal displacement after surgical treatment for idiopathic macular hole (IMH) with different internal limiting membrane (ILM) peeling patterns. METHODS: Totally 22 eyes from 20 patients with IMH were randomly allocated into two groups, N-T group (11 eyes) and T-N group (11 eyes). For patients in N-T group, ILM was peeled off from nasal to temporal retina. For patients in T-N group, ILM was peeled off from temporal to nasal retina. Preoperative, postoperative 1, 3, and 6mo, autofluorescence fundus images were collected for manual measurement of distances of fixed nasal (N), temporal (T), superior (S), and inferior (I) retinal points (bifurcation or crossing of retinal vessels) around the macula to the optic disc (OD). These were respectively defined as N-OD, T-OD, S-OD, and I-OD. The retinal displacement, macular hole closure rate, and best corrected visual acuity (BCVA) were compared between the two groups after surgery. RESULTS: At postoperative 1, 3, and 6mo, the macula slipped toward the OD, manifested by the decreased T-OD, N-OD, S-OD, and I-OD (P<0.05). No significant difference was found in the T-OD, N-OD, S-OD, and I-OD between N-T group and T-N group. IMH closure rate was 100% both in N-T group and T-N group. There was no significant difference in BCVA between two groups (P<0.05). CONCLUSION: The macula slips toward the OD after successful macular hole surgery. The two different ILM peeling pattern show similar visual outcome and retinal displacement, which means ILM peeling directions are not the influencing factor of postoperative retinal displacement.

Role of microRNA-25 in high glucose cultured Müller glia.

AIM: To investigate the role of microRNA-25 (miR-25) in proliferation and apoptosis of retinal Müller glia (MG) under high glucose condition. METHODS: The purity of the cultured cells was verified by immunocytochemistry and flow cytometry using antibodies that specifically recognized MG. The expression level of miR-25 under normal and high glucose conditions were validated by quantitative reverse transcription polymerase chain reaction (RT-qPCR). miR-25 mimics and negative control were transfected into MG and multiple functional experiments including cell counting kit-8 assay, EDU assay, and flow cytometry were conducted to explore the effects of miR-25 on the proliferation and apoptosis of high glucose cultured MG (HGMG). RESULTS: Immunocytochemistry and flow cytometry confirmed the high purity of primary cultured MG. RT-PCR results showed that the expression level of miR-25 was significantly repressed in HGMG, while over-expression of miR-25 by miR-25 mimic markedly inhibited the high glucose induced cell apoptosis and promoted the proliferation of MG. CONCLUSION: The expression level of miR-25 is significantly downregulated in HGMG and its overexpression could attenuate the high glucose damages on MG by promoting proliferation and reducing apoptosis.

Endothelium-derived semaphorin 3G attenuates ischemic retinopathy by coordinating ß-catenin-dependent vascular remodeling.

Abnormal angiogenesis and regression of the diseased retinal vasculature are key processes associated with ischemic retinopathies, but the underlying mechanisms that regulate vascular remodeling remain poorly understood. Here, we confirmed the specific expression of semaphorin 3G (Sema3G) in retinal endothelial cells (ECs), which was required for vascular remodeling and the amelioration of ischemic retinopathy. We found that Sema3G was elevated in the vitreous fluid of patients with proliferative diabetic retinopathy (PDR) and in the neovascularization regression phase of oxygen-induced retinopathy (OIR). Endothelial-specific Sema3G knockout mice exhibited decreased vessel density and excessive matrix deposition in the retinal vasculature. Moreover, loss of Sema3G aggravated pathological angiogenesis in mice with OIR. Mechanistically, we demonstrated that HIF-2α directly regulated Sema3G transcription in ECs under hypoxia. Sema3G coordinated the functional interaction between ß-catenin and VE-cadherin by increasing ß-catenin stability in the endothelium through the neuropilin-2 (Nrp2)/PlexinD1 receptor. Furthermore, Sema3G supplementation enhanced healthy vascular network formation and promoted diseased vasculature regression during blood vessel remodeling. Overall, we deciphered the endothelium-derived Sema3G-dependent events involved in modulating physiological vascular remodeling and regression of pathological blood vessels for reparative vascular regeneration. Our findings shed light on the protective effect of Sema3G in ischemic retinopathies.

A novel xeno-free culture system for human retinal pigment epithelium cells.

AIM: To find out an animal-free, xeno-free culture method for human fetal retinal pigment epithelium (fRPE) cells aiming for cell-replacement therapy. METHODS: Human AB serum, knock-out serum replacement (KSR) and B27 were supplemented as a substitute of fetal bovine serum (FBS) in culture medium of human fRPE cells. Cell morphology was examined by light microscope and transmission electron microscope. Proliferation ability was detected by cell cycle analysis and examination of KI67 expression. Apoptosis was analyzed using FACS. The expression of RPE-specific markers was demonstrated by quantitative real-time polymerase chain reaction (qPCR), Western blot (WB) and immunocytochemistry. Paracrine function was determined using enzyme-linked immunosorbent assay method. RESULTS: Our results indicated that the optimum concentration of KSR was 15%, the optimum concentration of B27 was 2%, and the optimum concentration of human AB serum was 10%. fRPE cells cultured in 15% KSR and 2% B27 media showed repressed propagation and differentiation ability, and gradually lost epithelial morphology and RPE function. While fRPE cells cultured in 10% human AB serum media showed a typical cobblestone morphology with pigmentation, elevated proliferation ability, satisfying paracrine function and expressed RPE-specific markers. CONCLUSION: Our study indicates that culturing fRPE cells in 10% human AB serum-supplemented medium is more favorable compared with KSR, B27 and traditional FBS-supplemented mediums when fRPE cells are to be applied in cell-based therapy.

Evaluation of the safety and quality of day-case cataract surgery based on 4151 cases.

AIM: To evaluate the safety, quality and prospects of day-case cataract surgery performed in a Jiangsu public tertiary hospital. METHODS: The general and clinical data for patients who underwent day-case cataract surgery between August 1, 2014 and December 31, 2016 at this hospital were collected. The incidences of intraoperative and postoperative complications, preoperative and postoperative best-corrected visual acuities (BCVAs), delayed discharge rate, rate of unplanned re-admission to hospital, and patient satisfaction were analyzed. RESULTS: A total of 4151 patients received cataract phacoemulsification surgery to correct age-related, congenital, traumatic, or complicated cataracts. Of these, age-related cataracts were the most frequently occurring. Patient age ranged from 18 to 101y and the vast majority of patients were between 60 and 80 years old. Of the 4151 patients, 64.73% (2687/4151) had a systemic disease. The number of patients increased over the years, with the average number of patients per month being 90.4, 124.83, and 183.42 in 2014, 2015 and 2016, respectively. The average preoperative BCVA was 0.102±0.057 and average postoperative BCVAs at 1d, 1wk, and 1mo post-surgery were 0.453±0.264, 0.657±0.285, and 0.734±0.244, respectively. For intraoperative complications, 4.12% (171/4151) had posterior capsule rupture, 0.79% (33/4151) had iris or ciliary body injury, and 0.048% (2/4151) had suprachoroidal hemorrhage. For postoperative complications, 4.38% (182/4151) had cornea edema, 7.78% (323/4151) had intraocular hypertension, 0.096% (4/4151) had IOL toxicity syndrome, 0.28% (12/4151) had retained lens cortex, and 0.048% (2/4151) had hyphema. The delayed discharge rate was 0.82% (44/4151) and the unplanned re-admission to the hospital was 0 (0/4151). The patient satisfaction rate was 91.42% (3795/4151). CONCLUSION: Day-case cataract surgery is safe and effective with good prospects for development.

Optic disc and peripapillary changes by optic coherence tomography in high myopia.

Myopia, a worldwide condition, is a multifactorial disease resulting in many ocular complications. Early onset of myopia has a great tendency to develop high myopia and pathological myopia later in life. The pathophysiology and progression of myopia is still unclear. Owing to its involving in visual function, optic disc and peripapillary change in high myopia can't be neglected, and it may help in better understanding of the pathophysiology or mechanism of myopia progression. Recently, advanced imaging techniques have been developed, such as optical coherence tomography (OCT), allowing for better detecting of optic disc and peripapillary change. OCT is a high-resolution and noninvasive measurement for detection of ocular structure. Herein, we provide an updated review of optic disc and peripapillary change in OCT image, including its characteristics and clinical significance. We also propose some problems needed further investigation.

Phacoemulsification combined with transpupillary removal of silicone oil and intracapsular intraocular lens implantation.

AIM: To estimate the effectiveness of phacoemulsification and foldable intraocular lens (IOL) implantation combined with transpupillary silicone oil removal. METHODS: There were 168 eyes of 168 candidate patients with cataract and silicone oil-filled eyes recruited in our study. All of the patients received the intraocular silicone oil removal surgery by transpupillary drainage and cataract extraction by phacoemulsification. Then the IOL implantation were also performed through corneal incision. RESULTS: The surgery was successfully completed in all eyes. Best corrected visual acuity (BCVA) and postoperative complications were recorded in three months after surgery. There were 143 eyes with BCVA improved, otherwise 25 eyes remained stable at the last follow-up visit. The mean BCVA statistically improved from 20/400±0.02 to 20/100±0.15 (P<0.001) and mean postoperative IOP was 13.85±2.18 mm Hg (P=0.415). No intra-operative complications were reported. CONCLUSION: Phacoemulsification combined with transpupillary removal of silicone oil is a safe and simple effective method. In general, it enables quick recovery of visual acuity with less complication rate.

Advances in the treatment of retinal angiomatous proliferation / 国际眼科杂志(Guoji Yanke Zazhi)

·Retinal angiomatous proliferation ( RAP) , also known as"type 3 neovascularization", is a well - recognized variation of neovascular age - related macular degeneration ( nARMD ) . Neovascularization is the basic pathological characteristic. Current view on the origin of the neovascularization is the deep retinal capillaries. The main clinical features include retinal pigment epithelium detachment( PED) and reticular pseudodrusen. These two features have close relation to the retinal pigment epithelium ( RPE ) tear and geographic atrophy ( GA ) , respectively, which may ultimately result in severe irreversible visual impairment. The disease has a rapid natural course and the majority of patients in early stage can develop into vision loss within 6mo. However, classical therapeutic managements, such as laser therapy, have limited efficacy and poor prognosis. Recently, RAP has been further understood with the application of OCT angiography and other new technologies in diagnosing, staging and monitoring RAP. Varieties of research on intravitreal injection of anti -vascular endothelial growth factor ( VEGF ) treatment to RAP have also revealed its promising results and proved its safety as well as effectiveness. All these have provided new knowledge on choosing the optimal treatment regimen in clinical.

Anthrax lethal toxin suppresses high glucose induced VEGF over secretion through a post-translational mechanism.

AIM: To prove anthrax lethal toxin (LeTx) blocks the mitogen activated protein kinases (MAPKs) activation by degrading the MAPK/ERK kinases (MEKs) to suppress vascular endothelial growth factor (VEGF) secretion. METHODS: Human adult retinal pigmented epithelium (ARPE) cells were cultured and treated with normal glucose, high glucose or high glucose with LeTx for additional 24, 48 or 72h for viable cell count. Total RNA from the ARPE was isolated for reverse transcription polymerase chain reaction (RT-PCR). The conditioned medium of ARPE cells treated in different group for 48h was filtered and diluted to detect the concentration of VEGF by enzyme-linked immunosorbant assays. Evaluate the role of MEK/MAPK pathway in the secretion of VEGF by immunoblotting. RESULTS: In this study, we proved high glucose induced activation of the MAPK extracellular signal-regulated kinase (ERK1/2) and p38 in the ARPE cell line was blocked by anthrax LeTx. LeTx also inhibited high glucose induced ARPE cell over proliferation. CONCLUSION: LeTx suppressed high glucose induced VEGF over secretion in the ARPE cells, mainly through a post-translational mechanism.

Aquaporin-4 knockout exacerbates corticosterone-induced depression by inhibiting astrocyte function and hippocampal neurogenesis.

AIMS: The predominant expression of aquaporin-4 (AQP4) in the brain implies that this water channel may be involved in a range of brain disorders. This study was designed to investigate the role of AQP4 in the pathogenesis of depression, and related possible biological mechanism. METHODS AND RESULTS: Wild-type (AQP4(+/+) ) and AQP4 knockout (AQP4(-/-) ) mice were given daily subcutaneous injections of corticosterone (20 mg/kg) for consecutive 21 days. Forced swimming test (FST) and tail suspension test (TST) showed longer immobility times in corticosterone-treated AQP4(-/-) genotype, indicating AQP4 knockout exacerbated depressive-like behaviors in mice. Using immunohistological staining, western blot, and enzyme-linked immunosorbent assay (ELISA), we found a significant loss of astrocytes, aggravated downregulation of excitatory amino acid transporter 2 (EAAT2), synapsin-1, and glial cell line-derived neurotrophic factor (GDNF) in the hippocampus of AQP4(-/-) mice. Moreover, even less hippocampal neurogenesis was identified in corticosterone-treated AQP4(-/-) mice in vivo and hippocampus-derived adult neural stem cells (ANSCs) in vitro. CONCLUSIONS: The present findings suggest AQP4 involves the pathogenesis of depression by modulating astrocytic function and adult neurogenesis, highlighting a novel profile of AQP4 as a potential target for the treatment for depression.

Interleukin-6 prevents NMDA-induced neuronal death via Gp130 signaling-dependent IP3R inhibition.

OBJECTIVE: To reveal the involvement of inositol 1,4,5-trisphosphate receptors (IP3R) and ryanodine receptors (RyR) in IL-6 prevention from neuronal apoptosis and necrosis induced by N-methyl-D-aspartate (NMDA). METHODS: Cerebellar granule neurons (CGNs) from 8-day-old rats were exposed to IL-6 for 8 days and then stimulated with NMDA for 30 min. The 2-aminoethoxydiphenyl borate (2-APB) and dantrolene (DAN) were used to antagonize IP3R and RyR, respectively. Anti-gp130 monoclonal antibody (mAb) was employed to neutralize gp130, a 130-kDa signal-transducing ß-subunit of IL-6 receptor complex. Neuronal apoptosis and necrosis were determined by TUNEL, fluorometric caspase-3 enzyme activity, annexin V-FITC/PI staining and ELISA. Western blot and real-time PCR measured IP3R1 and RyR2 expression, respectively. RESULTS: IL-6 prevented the elevation of TUNEL-positive cells and caspase-3 expression and activity, and also suppressed the increase in annexin V-FITC/PI-positive cells and DNA- and histone-associated nucleosomes in cultured CGNs evoked by NMDA. These anti-apoptotic and anti-necrotic effects of IL-6 were larger on DAN-treated cells than on 2-APB-exposed neurons, since 2-APB treatment alone significantly inhibited the neuronal apoptosis and necrosis but DAN exposure alone did not alter the apoptosis and necrosis induced by NMDA. In support of these results, IL-6 downregulated IP3R1 but did not affect RyR2 expression. All these IL-6 effects were blocked by anti-gp130 mAb. CONCLUSION: IL-6 prevention from NMDA-triggered Ca2+-induced Ca2+ release-mediated apoptosis and necrosis in CGNs depends on the inhibition of IP3R channel opening and expression rather than on RyR activity. IL-6 receptor-coupled gp130 signaling mediates this neuroprotection of IL-6 resistance to neuronal apoptosis and necrosis.

[Targeted sequencing identifies a hotspot mutation SNRNP200 p.S1087L correlates with novel phenotypes in retinitis pigmentosa].

OBJECTIVE: To identify the pathogenic mutation in a four-generation Chinese family with autosomal dominant retinitis pigmentosa (ADRP) and to analyze its associated clinical phenotypes. METHODS: Twelve participants from the index family were recruited, including 5 patients, 6 asymptomatic siblings, and one spouse. All participants underwent ophthalmic examinations, including best-corrected visual acuity (BCVA), visual field (VF) testing, fundus photography, and full-field flash electroretinography (ERG). Targeted sequence capture array technique with next-generation of high throughput sequencing(NGS) was performed to detect variants in 189 hereditary retinal disease (HRD) related genes, comprising 179 identified HRD-causing genes and 10 potential causative genes which were involved in pre-messenger RNA(pre-mRNA) splicing. Variants detected by targeted sequencing were filtered by bioinformatic analyses, validated by Sanger sequencing and intra-familiar analysis.Genotype-phenotype correlation was also analyzed. RESULTS: SNRNP200 p.S1087L was identified as the disease causative mutation for this family by targeted sequencing and optimized bioinformatic analyses. This family demonstrated early onset of the disease by presenting nyctalopia among 6 to 8 years old, performed rapid disease progression and severely impaired visual function by displaying loss of VF among 14 to 17 years old and decreased central vision among 21 to 28 years old. The fundus presentations and ERG results showed typical RP presentations. CONCLUSIONS: SNRNP200 p.S1087L is identified as a hotspot mutation but correlates with distinct phenotypes in the present family, including early onset of the disease, rapid disease progression, and severely impaired visual function. This study also give evidence to that molecular diagnostic platform for HRD can improve the detection rate of causative genes/mutations in HRD patients, thus providing important approaches for further investigation of the genetic causes for HRD.

[Gene mapping in a Chinese family with autosomal dominant centralpuiverulent cataract].

OBJECTIVE: Mapping the mutation gene for a Chinese family with autosomal dominant cataract. METHODS: It was a retrospective study. Thirty-two individuals in this family, including fifteen patients, eight normal siblings and nine spouses, were investigated and 8 ml blood was collected from each member under informed consent. Genomic DNA of all 29 members was isolated by standard protocol. A genome wide scan was performed after PCR amplification for microsatellite makers on autosomal chromosomes. LOD score was calculated by Linkage 5. 1 and GeneHunter software. RESULTS: Positive Lod score were obtained in 10 microsatellite makers (D20S186, D20S163, D20S915, D20S152, D20S98, D20S904, D20S875, D20S112, D20S1140, D20S432) on chromosome 20q, and the maximum LOD score with D20S904 was 6.02. CONCLUSIONS: Haplotype construction and multipoint analysis mapped the mutation gene in this inherited cataract family to the chromosome 20p12. 1-20p11.23 region between D20S186 and D20S912, which is an approximately 5.47 centimorgan length. This is the second congenital cataract locus linked to chromosome 20q.

A locus for autosomal dominant accessory auricular anomaly maps to 14q11.2-q12.

Accessory auricular anomaly is a small excrescence of skin that contains elastic cartilage on different regions of the helix and the face. Previous work has shown that the genetic trait of some patients with the isolated symptom of accessory auricular anomaly is autosomal dominant. To map the gene for autosomal dominant accessory auricular anomaly (ADAAA), we investigated a Chinese family with 11 affected individuals. We performed linkage analysis with microsatellite markers spanning the whole human-genome in the family. The inheritance pattern of the ADAAA family was autosomal dominant with complete penetrance. Two-point linkage analysis revealed significant maximum LOD scores of 4.20(D14S990 and D14S264, sita = 0) in the family. Haplotype construction and multipoint linkage analysis also confirmed the locus and defined the isolated ADAAA locus to a 9.84 cM interval between the markers D14S283 and D14S297. Our study assigned an isolated ADAAA locus to 14q11.2-q12. This is the first ADAAA locus reported to date.

[Cloning and eukaryotic expression of wild type and truncated mouse ciliary neurotrophic factor gene].

OBJECTIVE: To clone and eukaryotic express wild type and truncated mouse ciliary neurotrophic factor (CNTF) gene, and to observe the biological effect of two types of CNTF gene expressing in ARPE-19 cells. METHODS: RT-PCR was used to amplify the cDNA of CNTF gene, and truncated CNTF cDNA was obtained by site-directed mutagenesis. The two types of CNTF gene were cloned into plasmid pTracer-CMV and transfected to ARPE-19 cells. Dot blotting was used to detect the expression of CNTF. MTT and flow cytometry apoptosis assay were performed to observe the biological effect of CNTF expressing in ARPE-19 cells. RESULTS: Wild type and truncated CNTF gene were amplified by RT-PCR, and their eukaryotic expression plasmids were successfully constructed. After ARPE-19 cells transfected with two types of recombinant plasmids, the CNTF were detected in the supernatant of cells culture. MTT result shows that two types of CNTF have no proliferation promoting effect to ARPE-19 cells, and quantitive apoptosis assay implicated that CNTF could partially suppress the apoptosis that induced by the cells culturing with serum free culture. CONCLUSION: Expression of two types of CNTF in ARPE-19 cells gets prepared for gene therapy research of retinitis pigmentosa.