Detection of novel germline mutations in six breast cancer predisposition genes by targeted next-generation sequencing.

In this study, a customized amplicon-based target sequencing panel was designed to enrich the whole exon regions of six genes associated with the risk of breast cancer. Targeted next-generation sequencing (NGS) was performed for 146 breast cancer patients (BC), 71 healthy women with a family history of breast cancer (high risk), and 55 healthy women without a family history of cancer (control). Sixteen possible disease-causing mutations on four genes were identified in 20 samples. The percentages of possible disease-causing mutation carriers in the BC group (8.9%) and in the high-risk group (8.5%) were higher than that in the control group (1.8%). The BRCA1 possible disease-causing mutation group had a higher prevalence in family history and triple-negative breast cancer, while the BRCA2 possible disease-causing mutation group was younger and more likely to develop axillary lymph node metastasis (P < 0.05). Among the 146 patients, 47 with a family history of breast cancer were also sequenced with another 14 moderate-risk genes. Three additional possible disease-causing mutations were found on PALB2, CHEK2, and PMS2 genes, respectively. The results demonstrate that the six-gene targeted NGS panel may provide an approach to assess the genetic risk of breast cancer and predict the clinical prognosis of breast cancer patients.

Clinical Next Generation Sequencing for Precision Medicine in Cancer.

Rapid adoption of next generation sequencing (NGS) in genomic medicine has been driven by low cost, high throughput sequencing and rapid advances in our understanding of the genetic bases of human diseases. Today, the NGS method has dominated sequencing space in genomic research, and quickly entered clinical practice. Because unique features of NGS perfectly meet the clinical reality (need to do more with less), the NGS technology is becoming a driving force to realize the dream of precision medicine. This article describes the strengths of NGS, NGS panels used in precision medicine, current applications of NGS in cytology, and its challenges and future directions for routine clinical use.

Exon-level expression profiling of ocular tissues.

The normal gene expression profiles of the tissues in the eye are a valuable resource for considering genes likely to be involved with disease processes. We profiled gene expression in ten ocular tissues from human donor eyes using Affymetrix Human Exon 1.0 ST arrays. Ten different tissues were obtained from six different individuals and RNA was pooled. The tissues included: retina, optic nerve head (ONH), optic nerve (ON), ciliary body (CB), trabecular meshwork (TM), sclera, lens, cornea, choroid/retinal pigment epithelium (RPE) and iris. Expression values were compared with publically available Expressed Sequence Tag (EST) and RNA-sequencing resources. Known tissue-specific genes were examined and they demonstrated correspondence of expression with the representative ocular tissues. The estimated gene and exon level abundances are available online at the Ocular Tissue Database.

Increased expression of the WNT antagonist sFRP-1 in glaucoma elevates intraocular pressure.

Elevated intraocular pressure (IOP) is the principal risk factor for glaucoma and results from excessive impedance of the fluid outflow from the eye. This abnormality likely originates from outflow pathway tissues such as the trabecular meshwork (TM), but the associated molecular etiology is poorly understood. We discovered what we believe to be a novel role for secreted frizzled-related protein-1 (sFRP-1), an antagonist of Wnt signaling, in regulating IOP. sFRP1 was overexpressed in human glaucomatous TM cells. Genes involved in the Wnt signaling pathway were expressed in cultured TM cells and human TM tissues. Addition of recombinant sFRP-1 to ex vivo perfusion-cultured human eyes decreased outflow facility, concomitant with reduced levels of beta-catenin, the Wnt signaling mediator, in the TM. Intravitreal injection of an adenoviral vector encoding sFRP1 in mice produced a titer-dependent increase in IOP. Five days after vector injection, IOP increased 2 fold, which was significantly reduced by topical ocular administration of an inhibitor of a downstream suppressor of Wnt signaling. Thus, these data indicate that increased expression of sFRP1 in the TM appears to be responsible for elevated IOP in glaucoma and restoring Wnt signaling in the TM may be a novel disease intervention strategy for treating glaucoma.

Increased expression of serum amyloid A in glaucoma and its effect on intraocular pressure.

PURPOSE: To search for and validate potential molecular pathogenic mechanisms in the trabecular meshwork (TM) responsible for the elevated intraocular pressure (IOP) associated with glaucoma. METHODS: Gene chip arrays were used to identify differential gene expression in glaucomatous TM tissues. Serum amyloid A (SAA) upregulation was subsequently confirmed with quantitative PCR (QPCR) and ELISA. The effect of SAA on gene expression of cultured human TM cells was tested with gene chip arrays and verified with ELISA, and its effect on IOP was evaluated in the human ocular perfusion organ culture. RESULTS: Microarray analysis showed that the expression of SAA2 was increased in TM tissues from donors with glaucoma. This finding was subsequently confirmed by QPCR. The SAA mRNA levels were increased in glaucoma TM tissues by more than 5-fold (P < 0.05) and in cultured TM cells derived from donors with glaucoma by 25-fold (P < 0.05) compared with controls. SAA protein levels in the TM of glaucoma patients were also significantly (P < 0.05) elevated by 2.9-fold. Treatment of cultured human TM cells with recombinant SAA affected gene expression, including a 22-fold up-regulation of interleukin-8 (P < 0.001). SAA increased IOP by approximately 40% (P < 0.05) in the human ocular perfusion organ culture without any observable changes in the morphology of the tissues involved in aqueous outflow. CONCLUSIONS: These findings indicate that SAA, which is an acute-phase apolipoprotein that plays important roles in infection, inflammation, and tissue repair, may contribute to the pathogenic changes to the TM in glaucoma.

Effect of immunomodulation with anti-CD40L antibody on adenoviral-mediated transgene expression in mouse anterior segment.

PURPOSE: Gene transduction using adenoviral vectors is an important research tool. To assess and optimize this technique for glaucoma research, we characterized green fluorescent protein (GFP) expression in the mouse eye after intraocular injection of adenoviral vector encoding GFP (Ad5.CMV-GFP) and evaluated the effect of anti-CD40L antibody administration on GFP expression. METHODS: Mice were injected with Ad5.CMV-GFP intracamerally (IC) or intravitreally (IVT) with or without anti-CD40L antibody treatment. GFP expression was assessed by in vivo fluorescence intensity with a standardized grading scale. Location of expression was analyzed histologically by fluorescence microscopy. RESULTS: Intraocular injection of Ad5.CMV-GFP induced titer-dependent expression of GFP in the anterior segment. In vivo fluorescence was detectable but low after IC injection. After IVT injection, fluorescence in the eye peaked at days 4-7 with a fluorescence grade of 3.0 +/-0.0 (mean +/-SEM, n=6; injection with 1x10(8) pfu vector). After day 7, GFP expression declined significantly. Treatment with anti-CD40L antibody increased fluorescence intensity after IC injection, and prolonged GFP expression in the IVT group. At day 43, fluorescence grades of the IVT group were 2.8 +/-0.7 (with anti-CD40L) and 1.2 +/-0.6 (without antibody). Three-Way ANOVA confirmed that GFP expression was significantly higher in the anti-CD40L than the no antibody group (p=0.013), significantly higher in the IVT than the IC group (p=0.003), and significantly higher in the high viral titer (1x10(8) pfu) than the low titer (1x10(7) pfu) group (p=0.010). Fluorescence microscopic examination of cross-sections of eyes indicated GFP expression in the trabecular meshwork (TM), corneal endothelium, and sporadically iris, ciliary body and lens epithelium. CONCLUSIONS: Intraocular injection of Ad5.CMV-GFP induced GFP expression in the mouse anterior segment, including the TM. Expression was more prominent after IVT injection than IC injection. Anti-CD40L antibody treatment increased both intensity and duration of GFP expression. These findings provide important and practical means to improve duration and efficiency of adenovirus-mediated transgene expression in the eye.

[Intraocular pressure measurement in rodents].

The ability to measure intraocular pressure in rodents is very important for the advancement of glaucoma research. This review article describes various currently used methods, such as, microcannulation, servo-null micropipette, Tonopen tonometer, rebound tonometer, Goldmann applanation tonometer, Schiötz indentation tonometer, pneumatonometer, and optic interferometry tonometer. Their principles of operation, advantages and limitations, as well as a calibration method are also discussed.

Noninvasive measurement of rodent intraocular pressure with a rebound tonometer.

PURPOSE: The present study evaluated the applicability of a rebound tonometer in measuring intraocular pressure (IOP) in rats and mice. METHODS: The accuracy of the TonoLab rebound tonometer was determined in cannulated mouse and rat eyes. IOP was manipulated by changing reservoir height, and tonometer pressure readings were recorded by an independent observer. IOP values were recorded in conscious Wistar rats and in four different strains of mice. The effects of anesthesia on IOP were evaluated in two different strains of mice. RESULTS: The IOP readings generated by the rebound tonometer correlated very well with the actual pressure in the eye. In rats, this linear correlation had a slope of 0.96 +/- 0.05 (mean +/- SEM, n = 4) and a Y-intercept of -2.1 +/- 1.2. In mice, the slope was 0.99 +/- 0.05 (n = 3), and the Y-intercept was 0.8 +/- 1.4. Using this method, the resting IOP of conscious male Wistar rats was observed to be 18.4 +/- 0.1 mm Hg (n = 132). In mice, strain differences in IOP were detected. Baseline IOP values in Balb/c, C57-BL/6, CBA, and 11- to 12-month-old DBA/2J mice were 10.6 +/- 0.6, 13.3 +/- 0.3, 16.4 +/- 0.3, and 19.3 +/- 0.4 mm Hg (n = 12), respectively. In separated studies, anesthesia lowered IOP from 14.3 +/- 0.9 to 9.2 +/- 0.5 mm Hg (n = 8) in C57-BL/6 mice, and from 16.6 +/- 0.4 to 9.4 +/- 0.6 mm Hg (n = 10) in CBA mice. CONCLUSIONS: The rebound tonometer was easy to use and accurately measured IOP in rats and mice. This technique, together with advances in genetic and other biological studies in rodents, will be valuable in the further understanding of the etiology and pathology of glaucoma.

Microbead-induced ocular hypertensive mouse model for screening and testing of aqueous production suppressants for glaucoma.

PURPOSE: To characterize the microbead-induced ocular hypertension (OHT) mouse model and investigate its potential use for preclinical screening and evaluation of ocular hypotensive agents, we tested the model's responses to major antiglaucoma drugs. METHODS: Adult C57BL/6J mice were induced to develop OHT unilaterally by intracameral injection of microbeads. The effects of the most commonly used ocular hypotensive drugs, including timolol, brimonidine, brinzolamide, pilocarpine, and latanoprost, on IOP and glaucomatous neural damage were evaluated. Degeneration of retinal ganglion cells (RGCs) and optic nerve axons were quantitatively assessed using immunofluorescence labeling and histochemistry. Thickness of the ganglion cell complex (GCC) was also assessed with spectral-domain optical coherence tomography (SD-OCT). RESULTS: A microbead-induced OHT model promptly responded to drugs, such as timolol, brimonidine, and brinzolamide, that lower IOP through suppressing aqueous humor production and showed improved RGC and axon survival as compared to vehicle controls. Accordingly, SD-OCT detected significantly less reduction of GCC thickness in mice treated with all three aqueous production suppressants as compared to the vehicle contol-treated group. In contrast, drugs that increase aqueous outflow, such as pilocarpine and latanoprost, failed to decrease IOP in the microbead-induced OHT mice. CONCLUSIONS: Microbead-induced OHT mice carry dysfunctional aqueous outflow facility and therefore offer a unique model that allows selective screening of aqueous production suppressant antiglaucoma drugs or for studying the mechanisms regulating aqueous humor production. Our data set the stage for using GCC thickness assessed by SD-OCT as an imaging biomarker for noninvasive tracking of neuronal benefits of glaucoma therapy in this model.

Existence of the canonical Wnt signaling pathway in the human trabecular meshwork.

PURPOSE: We previously discovered elevated levels of secreted frizzled-related protein 1 (sFRP1), the Wnt signaling pathway inhibitor, in the glaucomatous trabecular meshwork (GTM), and found that key canonical Wnt signaling pathway genes are expressed in the trabecular meshwork (TM). The purpose of our study was to determine whether a functional canonical Wnt signaling pathway exists in the human TM (HTM). METHODS: Western immunoblotting and/or immunofluorescent microscopy were used to study ß-catenin translocation as well as the actin cytoskeleton in transformed and primary HTM cells. A TCF/LEF luciferase assay was used to study functional canonical Wnt signaling, which was confirmed further by WNT3a-induced expression of a pathway target gene, AXIN2, via quantitative PCR. Intravitreal injection of an Ad5 adenovirus expressing Dickkopf-related protein-1 (DKK1) was used to study the in vivo effect of canonical Wnt signaling on IOP in mice. RESULTS: WNT3a induced ß-catenin translocation in the HTM, which was blocked by co-treatment with sFRP1. Similarly, WNT3a enhanced luciferase levels in TCF/LEF luciferase assays, which also were blocked by sFRP1. Furthermore, AXIN2 expression was elevated significantly by WNT3a. However, neither WNT3a nor sFRP1 affected actin cytoskeleton organization, which theoretically could be regulated by noncanonical Wnt signaling in HTM cells. Exogenous DKK1, a specific inhibitor for the canonical Wnt signaling pathway, or sFRP1 elevated mouse IOP to equivalent levels. CONCLUSIONS: There is a canonical Wnt signaling pathway in the TM, and this canonical Wnt pathway, but not the noncanonical Wnt signaling pathway, regulates IOP.

Adenoviral gene transfer of active human transforming growth factor-{beta}2 elevates intraocular pressure and reduces outflow facility in rodent eyes.

Purpose. Glaucoma is a leading cause worldwide of blindness and visual impairment. Transforming growth factor-beta2 (TGFbeta2) has been implicated in the pathogenesis of primary open-angle glaucoma (POAG) based on elevated levels in glaucomatous aqueous humor and its ability to induce extracellular matrix (ECM) remodeling in the trabecular meshwork (TM). The goal of this study was to generate a rodent model of POAG using viral gene transfer of human TGFbeta2. Methods. Latent (hTGFbeta2(WT)) or active (C226S, C228S; hTGFbeta2(226/228)) TGFbeta2-encoding cDNA was cloned into the pac.Ad5.CMV.K-N.pA shuttle vector for generation of replication-deficient adenovirus. Empty adenovirus (Ad5.CMV.K-N.pA) was used as a control. Adenoviral expression of active and total TGFbeta2 was assayed in vitro by the transduction of Chinese hamster ovary and trabecular meshwork cells. BALB/cJ mice or Wistar rats were injected either intracamerally or intravitreally with the adenovectors and assessed for changes in intraocular pressure (IOP) using the rebound tonometer. At peak IOP, aqueous outflow facility and total TGFbeta2 levels in aqueous humor were measured. Mouse eye morphology was assessed by hematoxylin and eosin staining. Results. Adenoviral gene transfer of hTGFbeta2(226/228), but not hTGFbeta2(WT), to the rodent eye elevated IOP in rat (43%, P < 0.001) and mouse (110%, P < 0.001) and reduced aqueous humor outflow facility in the mouse. The TGFbeta2-induced ocular hypertension correlated with anterior segment TGFbeta2 expression levels (P < 0.0001). Conclusions. The adenoviral TGFbeta2 rodent model displays the glaucoma risk factors of elevated IOP and decreased aqueous outflow facility and may potentially serve as a model for studying glaucoma.

Acute effects of glaucoma medications on rat intraocular pressure.

The rat has been used increasingly in glaucoma research, but many aspects regarding the regulation of its intraocular pressure (IOP) are still unknown. For example, it is not clear whether glaucoma medications can lower IOP in the rat similarly to human. This information will be valuable in evaluating this animal model for its usefulness in predicting drug effects in patients. Hence, we tested the acute IOP effects of selected glaucoma drugs topical administered onto the rat eye. In these studies, IOP was measured using the Tono-Pen XL tonometer. After a correlation between the IOP reported by the Tono-Pen and actual IOP was established, IOP measurements were obtained in slightly sedated adult rats. Effects of glaucoma medications were tested in two groups of animals. One group (12 h/L) was housed in a 12-h/12-h light/dark cycle. The other (24 h/L) was housed under constant light. Exposure of the animals to constant light increased their basal IOP from 20.5+/-0.6 mmHg (mean+/-S.E.M., n=12) to 32.0+/-0.5 mmHg. At 3 h after topical administration, Betoptic S lowered IOP by 4.3+/-1.7 mmHg (n=6) and 3.7+/-0.3 mmHg (n=6) in the 12 and 24h/L rats, respectively. Pilocarpine did not affect rat IOP. Xalatan produced a biphasic response in the rat. At 3h after topical administration, it increased IOP by 7.9+/-1.4 and 7.0+/-1.0 mmHg in the 12 and 24 h/L rats, respectively. By the next day, it decreased IOP by 3.0+/-1.0 and 6.0+/-0.8 mmHg in the 12 and 24 h/L rats, respectively. The IOP-enhancing effect of Xalatan was dose-dependent. The present study indicates that IOP responses of the rat to different pharmacological agents are not identical to those of the human. In the rat, Betoptic S, but not pilocarpine, lowered IOP. Xalatan initially increased then decreased IOP.