Identification of a novel NHS mutation in a Chinese family with Nance-Horan syndrome.

PURPOSE: To identiy the disease causing mutation in a Chinese family presenting with early-onset cataract and dental anomalies. MATERIALS AND METHODS: A specific Hereditary Eye Disease Enrichment Panel (HEDEP) (personalized customization by MyGenostics, Baltimore, MD) based on targeted exome capture technology was used to collect the protein coding regions of 30 early-onset cataract associated genes, and high throughput sequencing was done with Illumina HiSeq 2000 platform. The identified variant was confirmed with Sanger sequencing. RESULTS: A novel deletion in exon 4 (c.852delG) of NHS gene was identified; the identified 1 bp deletion altered the reading frame and was predicted to result in a premature stop codon after the addition of twelve novel amino acid (p.S285PfsX13). This mutation co-segregated in affected males and obligate female carriers, but was absent in 100 matched controls. CONCLUSIONS: Our findings broaden the spectrum of NHS mutations causing Nance-Horan syndrome and phenotypic spectrum of the disease in Chinese patients.

Novel mutations of CRB1 in Chinese families presenting with retinal dystrophies.

PURPOSE: To identify disease-causing mutations in Chinese families who presented with retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA). METHODS: The pathogenic variant in a Chinese family with autosomal dominant RP was investigated with a specific hereditary eye disease enrichment panel (HEDEP) based on targeted exome capture technology. The identified variant was confirmed with Sanger sequencing. CRB1 mutations in 67 patients with sporadic retinal dystrophy were examined with Sanger sequencing. RESULTS: Compound heterozygous mutations were identified in a family who had undergone HEDEP analysis. After complete sequence analysis of the CRB1 gene was performed in 67 patients with sporadic retinal dystrophy, other compound heterozygous mutations were detected in three families. The mutations included three novel heterozygous mutations: c.3059delT (p.M1020SfsX1), c.3460T>A (p.C1154S), and c.4207G>C (p.E1403Q). The mutation frequency of CRB1 in this study was 5.9% (8/136). CONCLUSIONS: Our findings broaden the spectrum of CRB1 mutations and the phenotypic spectrum of the disease in Chinese patients. The results from this study show that patients with LCA carry CRB1 null mutations more frequently than patients with RP.

Novel mutations of RPGR in Chinese retinitis pigmentosa patients and the genotype-phenotype correlation.

X-linked Retinitis Pigmentosa (XLRP) accounts for 10-20% of all RP cases, and represents the most severe subtype of this disease. Mutations in the Retinitis Pigmentosa GTPase Regulator (RPGR) gene are the most common causes of XLRP, accounting for over 70-75% of all XLRP cases. In this work, we analyzed all the exons of RPGR gene with Sanger sequencing in seven Chinese XLRP families, two of these with a provisional diagnosis of adRP but without male-to-male transmission. Three novel deletions (c.2233_34delAG; c.2236_37delGA and c.2403_04delAG) and two known nonsense mutations (c.851C→G and c.2260G→T) were identified in five families. Two novel deletions (c.2233_34delAG and c.2236_37delGA) resulted in the same frame shift (p.E746RfsX22), created similar phenotype in Family 3 and 4. The novel deletion (c.2403_04delAG; p.E802GfsX31) resulted in both XLRP and x-linked cone-rod dystrophy within the male patients of family 5, which suggested the presence of either genetic or environmental modifiers, or both, play a substantial role in disease expression. Genotype-phenotype correlation analysis suggested that (1) both patients and female carriers with mutation in Exon 8 (Family 1) manifest more severe disease than did those with ORF15 mutations (Family 2&3&4); (2) mutation close to downstream of ORF15 (Family 5) demonstrate the early preferential loss of cone function with moderate loss of rod function.

Targeted exome capture and sequencing identifies novel PRPF31 mutations in autosomal dominant retinitis pigmentosa in Chinese families.

OBJECTIVES: To identify disease-causing mutations in two Chinese families with autosomal dominant retinitis pigmentosa (adRP). DESIGN: Prospective analysis. PATIENTS: Two Chinese adRP families underwent genetic diagnosis. A specific hereditary eye disease enrichment panel (HEDEP) based on targeted exome capture technology was used to collect the protein coding regions of targeted 371 hereditary eye disease genes; high throughput sequencing was done with the Illumina HiSeq 2000 platform. The identified variants were confirmed with Sanger sequencing. SETTING: All experiments were performed in a large laboratory specialising in genetic studies in the Department of Ophthalmology, Peking University Third Hospital. RESULTS: Two novel mutations, including one splice site mutation (Int10 c.1074-2 A>T; p.Y359SfsX29) and one insertion (c.824_825insA; p.Y275X) of PRPF31 were identified in the two families. The two mutations segregated with the disease phenotype in their respective families. CONCLUSIONS: Our findings broaden the spectrum of PRPF31 mutations causing adRP and the phenotypic spectrum of the disease in Chinese patients. The HEDEP based on targeted exome capture technology is an efficient method for molecular diagnosis in adRP patients.

Role of endoplasmic reticulum stress in the loss of retinal ganglion cells in diabetic retinopathy.

Endoplasmic reticulum stress is closely involved in the early stage of diabetic retinopathy. In the present study, a streptozotocin-induced diabetic animal model was given an intraperitoneal injection of tauroursodeoxycholic acid. Results from immunofluorescent co-localization experiments showed that both caspase-12 protein and c-Jun N-terminal kinase 1 phosphorylation levels significantly in-creased, which was associated with retinal ganglion cell death in diabetic retinas. The C/ERB mologous protein pathway directly contributed to glial reactivity, and was subsequently responsible for neuronal loss and vascular abnormalities in diabetic retinopathy. Our experimental findings dicate that endoplasmic reticulum stress plays an important role in diabetes-induced retinal neu-ronal loss and vascular abnormalities, and that inhibiting the activation of the endoplasmic reticulum stress pathway provides effective protection against diabetic retinopathy.

Immunoglobulin gene locus events in epithelial cells of lactating mouse mammary glands.

It has been believed that the immunoglobulin (Ig) found abundantly in the colostrum of lactating mammalian is derived from serum or secreted by plasma cells present in the mammary gland. The recent finding of Ig gene rearrangements in breast cancer cells and benign hyperplastic breast epithelial cells suggests that it is likely that hyperplastic mammary gland epithelial cells during lactation can also produce Ig. In this study, we have demonstrated the presence of abundant amounts of Ig heavy and light chain transcripts in sorted cytokeratin 18-positive mammary gland epithelial cells of lactating mice. Interestingly, we found two specific Igkappa variable region sequences (V(CW9)J(kappa1) and V(BV9)J(kappa1)) that were dominantly expressed in different strains of mice. Our data demonstrate that IgG is expressed by mammary gland epithelial cells of lactating mice, and suggest that the IgG found in murine colostrum is at least partially produced by the mammary gland epithelial cells.

Baicalein reduces inflammatory process in a rodent model of diabetic retinopathy.

PURPOSE: This study was designed to elucidate the role of inflammatory process in diabetic retinopathy and to investigate the effect of baicalein treatment on diabetic rat. METHODS: Retinal microglial cells were identified with CD11b antibody, and retinal Müller cells were identified with glial fibrillary acidic protein (GFAP). The gene expression of interleukin (IL)-18, tumor necrosis factor (TNF)-alpha, and IL-1beta was examined by quantitative real-time PCR. The expression of GFAP and vascular endothelial growth factor (VEGF) was examined by quantitative real-time PCR, immunohistochemistry, and Western blot analysis. Vascular permeability was measured in vivo by bovine serum albumin conjugated with FITC. Baicalein was given by oral administration (150 mg/kg/d) with an animal feeding needle beginning 5 days after streptozotocin (STZ) injection. RESULTS: By 24 weeks after onset of diabetes, microglial cells were activated and proliferated, and Müller cells upregulated their GFAP and VEGF expression. Pro-inflammatory factors, including IL-18, TNF-alpha, and IL-1beta, were significantly upregulated. Obvious vascular leakage and abnormality were demonstrated, and ganglion cell loss was significant. Baicalein treatment ameliorated diabetes-induced microglial activation and pro-inflammatory expression, reduced the GFAP and VEGF expression from Müller cells, and significantly reduced vascular abnormality and ganglion cell loss within the retina. CONCLUSIONS: Inflammatory process, characterized by microglial activation and Müller cells dysfunction, was implicated in STZ-induced diabetic retinopathy. Baicalein treatment ameliorated inflammatory process, and therefore inhibited vascular abnormality and neuron loss in diabetic retinas.

[Activation of endoplasmic reticulum stress proteins in light-induced retinal degeneration].

OBJECTIVE: To investigate the expression of endoplasmic reticulum stress proteins in photoreceptor apoptosis in light-induced retinal degeneration. METHODS: Exposure to excessive levels of light induced photoreceptor apoptosis and had been previously used as a model for the study of retinal degeneration. Photoreceptor apoptosis was detected by terminal dUTP transferase nick end labeling (TUNEL). The protein expression levels of ER stress sensors including glucosejregulated protein-78 (GRP78/BiP), caspase-12, phospho-eukaryotic initiation factor 2alpha (eIF2alpha) and phospho- double-stranded RNA-activated protein kinase-like endoplasmic reticulum kinase (PERK) were examined by immunojfluorescence and Western blot analysis. RESULTS: Following light exposure, the protein expression levels of GRP78/BiP, caspase-12, phospho-eIF2alpha and phospho-PERK were up-regulated in a time dependent manner. The up-regulation of these proteins coincided with or preceded the photoreceptor apoptosis. At the peak of their expression, they were mainly located in the photoreceptor inner segments and/or outer nuclear layers (ONL). CONCLUSION: Activation of endoplasmic reticulum stress proteins appears to play an important role in light-induced retinal degeneration. Therefore endoplasmic reticulum stress modulators could become a strong candidate for a therapeutic agent in treatment of these diseases.

Endoplasmic reticulum stress is activated in light-induced retinal degeneration.

Exposure to excessive levels of light induces photoreceptor apoptosis and has previously been used as a model for the study of retinal degeneration. During the light exposure, intracellular calcium levels increase, and reactive oxygen species (ROS) are generated, which have been shown to cause endoplasmic reticulum (ER) stress. In the present study, we investigated the role of ER stress in light-induced photoreceptor apoptosis. Our study demonstrated that, after light exposure, the ER stress sensors including glucose-regulated protein-78 (GRP78/BiP), caspase-12, phospho-eukaryotic initiation factor 2 alpha (eIF2 alpha), and phospho-pancreatic ER kinase (PERK) were significantly up-regulated in a time-dependent manner. The up-regulation of these proteins coincided with or preceded the photoreceptor apoptosis indicated by TUNEL. These data showed that ER stress played an important role in light-induced photoreceptor apoptosis. Therefore, ER stress modulators could be strong candidates as therapeutic agents in the treatment of retinal degenerative diseases.

[Endoplasmic reticulum stress proteins are activated in rd retinal degeneration].

OBJECTIVE: To investigate the expression of endoplasmic reticulum (ER) stress proteins in photoreceptor apoptosis in rd mouse (Pde6bRd1/Rd1). METHODS: Photoreceptor apoptosis in rd mouse was detected by terminal dUTP transferase nick end labeling (TUNEL). The protein expression of ER stress sensors including glucose-regulated protein-78 (GRP78/BiP), caspase-12, phospho-eukaryotic initiation factor 2alpha (eIF2alpha) and phospho-pancreatic ER kinase (PERK) was examined by immunofluorescence and Western Blot analysis. RESULTS: Accompanying photoreceptor apoptosis in rd mouse, protein expression of GRP78/BiP, caspase-12, phospho-eIF2alpha and phospho-PERK was up-regulated in a time dependent manner. The up-regulation of these proteins coincided with or preceded the photoreceptor apoptosis. At the peak of their expression, they were mainly located in the photoreceptor inner segment and/or outer nuclear layer (ONL). CONCLUSION: Activation of ER stress proteins appears to play an important role in rd retinal degeneration. Therefore endoplasmic reticulum stress modulators could be a strong candidate as a therapeutic agent in the treatment of these diseases.

Activation of endoplasmic reticulum stress in degenerating photoreceptors of the rd1 mouse.

PURPOSE: Endoplasmic reticulum (ER) stress has been implicated in a wide variety of neurodegenerative disorders of the central nervous system (CNS). This study was designed to elucidate the role of ER stress in photoreceptor apoptosis in the rd1 mouse. METHODS: Photoreceptor apoptosis in the rd1 mouse was detected by terminal dUTP transferase nick-end labeling (TUNEL). Protein expressions of ER stress sensors, including glucose-regulated protein-78 (GRP78/BiP), caspase-12, phospho-eukaryotic initiation factor 2alpha (eIF2alpha), and phospho-pancreatic ER kinase (PERK), were examined by immunofluorescence and Western blot assays. RESULTS: Accompanying photoreceptor apoptosis in the rd1 mouse, the protein expressions of GRP78/BiP, caspase-12, phospho-eIF2alpha, and phospho-PERK were upregulated in a time-dependent manner. The upregulation of these proteins coincided with or preceded photoreceptor apoptosis. At the peak of their expression, these proteins were primarily located in the photoreceptor inner segments, the outer nuclear layer, or both. CONCLUSIONS: ER stress plays an important role in photoreceptor apoptosis in the rd1 mouse. Therefore, ER stress modulators may be strong candidates as therapeutic agents in the treatment of retinal degenerative diseases.

Identification of sequential events and factors associated with microglial activation, migration, and cytotoxicity in retinal degeneration in rd mice.

PURPOSE: To elucidate the role of activated microglia in the photoreceptor apoptosis of rd mice by identifying sequential events and factors associated with microglial activation, migration, and cytotoxicity during retinal degeneration. METHODS: Photoreceptor apoptosis in rd mice at postnatal days (P)8, 10, 12, 14, 16, and 18 was detected by terminal dUTP transferase nick end labeling (TUNEL). Retinal microglia were identified by CD11b antibody. Expression of chemokine mRNA, including monocyte chemoattractant protein (MCP)-1, MCP-3, macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, regulated on activation normal T-cell expressed and secreted (RANTES), interferon-gamma-inducible 10-kDa protein (IP-10), and fractalkine in the retina were examined by reverse transcription-polymerase chain reaction (RT-PCR) assay. Production of tumor necrosis factor (TNF)-alpha in the dystrophic retina was studied by enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry analysis. Microglial expression of TNF-alpha was determined by double immunolabeling. RESULTS: Whereas photoreceptor apoptosis in the rd mice started at P10 and reached a peak at P16, activation and migration of microglial cells were observed at P10 and peaked at P14. The expression of MCP-1, MCP-3, MIP-1alpha, MIP-1beta, and RANTES transcripts were noted at P8 and reached a peak at P12. Production of TNF-alpha was noted in the outer nuclear layer (ONL) of the rd mice at P8 and reached a peak at P12. At the peak of microglial activity, TNF-alpha was predominantly expressed in the activated microglial cells in the ONL. CONCLUSIONS: Activation of microglia, as well as expression of their signaling molecules (chemokines) and microglia-derived toxic factor (TNF-alpha), coincides with or precedes the occurrence of photoreceptor apoptosis, suggesting activated microglia play a major role in retinal degeneration in rd mice. The chemokines MCP-1, MCP-3, MIP-1alpha, MIP-1beta, and RANTES are involved in activation and recruitment of the microglia to the degenerating photoreceptor cell layer. TNF-alpha, produced by the activated microglia, may accentuate the photoreceptor cell death.

Human epithelial cancers secrete immunoglobulin g with unidentified specificity to promote growth and survival of tumor cells.

Immunoglobulins (Igs) are found thus far only to be produced by differentiated B lymphocytes. By immunohistochemistry analysis, in situ hybridization, and laser capture microdissection-assisted single-cell PCR, we demonstrate that human cancers of epithelial origin, including carcinomas of breast, colon, liver, lung, established epithelial cancer lines, as well as some normal lung tissues, also produce IgG in both cytoplasmic and secreted forms. Furthermore, blockade of tumor-derived IgG by either antisense DNA or antihuman IgG antibody increased programmed cell death and inhibited growth of cancer cells in vitro. More importantly, administration of antihuman IgG antibody also suppressed the growth of an IgG-secreting carcinoma line in immunodeficient nude mice. Our results support a role of tumor-derived IgG as growth factor for epithelial cancers. Prevalent expression of IgG in human carcinomas and its growth-promoting functions may have important implications in growth regulation and targeted therapy of human cancers.