Sarah F. Janssen

Common Genetic Determinants of Intraocular Pressure and Primary Open-Angle Glaucoma

Intraocular pressure (IOP) is a highly heritable risk factor for primary open-angle glaucoma and is the only target for current glaucoma therapy. The genetic factors which determine IOP are largely unknown. We performed a genome-wide association study for IOP in 11,972 participants from 4 independent population-based studies in The Netherlands. We replicated our findings in 7,482 participants from 4 additional cohorts from the UK, Australia, Canada, and the Wellcome Trust Case-Control Consortium 2/Blue Mountains Eye Study. IOP was significantly associated with rs11656696, located in GAS7 at 17p13.1 (p = 1.4×10−8), and with rs7555523, located in TMCO1 at 1q24.1 (p = 1.6×10−8). In a meta-analysis of 4 case-control studies (total N = 1,432 glaucoma cases), both variants also showed evidence for association with glaucoma (p = 2.4×10−2 for rs11656696 and p = 9.1×10−4 for rs7555523). GAS7 and TMCO1 are highly expressed in the ciliary body and trabecular meshwork as well as in the lamina cribrosa, optic nerve, and retina. Both genes functionally interact with known glaucoma disease genes. These data suggest that we have identified two clinically relevant genes involved in IOP regulation.

Common genetic variants associated with open-angle glaucoma

Open-angle glaucoma (glaucoma) is a major eye disorder characterized by optic disc pathology. Recent genome-wide association studies identified new loci associated with clinically relevant optic disc parameters, such as the optic disc area and vertical cup-disc ratio (VCDR). We examined to what extent these loci are involved in glaucoma. The loci studied include ATOH7, CDC7/TGFBR3 and SALL1 for optic disc area, and CDKN2B, SIX1, SCYL1/LTBP3, CHEK2, ATOH7 and DCLK1 for VCDR. We performed a meta-analysis using data from six independent studies including: the Rotterdam Study (n= 5736), Genetic Research in Isolated Populations combined with Erasmus Rucphen Family study (n= 1750), Amsterdam Glaucoma Study (n= 296) and cohorts from Erlangen and Tübingen (n= 1363), Southampton (n= 702) and deCODE (n= 36 151) resulting in a total of 3161 glaucoma cases and 42 837 controls. Of the eight loci, we found significant evidence (P= 1.41 × 10(-8)) for the association of CDKN2B with glaucoma [odds ratio (OR) for those homozygous for the risk allele: 0.76; 95% confidence interval (CI): 0.70-0.84], for the role of ATOH7 (OR: 1.28; 95% CI: 1.12-1.47) and for SIX1 (OR: 1.20; 95% CI: 1.10-1.31) when adjusting for the number of tested loci. Furthermore, there was a borderline significant association of CDC7/TGFBR3 and SALL1 (both P= 0.04) with glaucoma. In conclusion, we found consistent evidence for three common variants (CDKN2B, ATOH7 and SIX1) significantly associated with glaucoma. These findings may shed new light on the pathophysiological protein pathways leading to glaucoma, and point to pathways involved in the growth and development of the optic nerve.

The vast complexity of primary open angle glaucoma : Disease genes, risks, molecular mechanisms and pathobiology

Primary open angle glaucoma (POAG) is a complex progressive optic nerve neuropathy triggered by both environmental and genetic risk factors. Several ocular tissues, including the ciliary body, trabecular meshwork and optic nerve head, and perhaps even brain tissues, are involved in a chain of pathological events leading to POAG. Genetic risk evidence for POAG came from family linkage-studies implicating a small number of disease genes (MYOC, OPTN, WDR36). Recent Genome Wide Association Studies (GWAS) identified a large number of new POAG loci and disease genes, such as CAV1, CDKN2B and GAS7. In the current study, we reviewed over 120 family and GWA studies. We selected in total 65 (candidate) POAG disease genes and proceeded to assess their function, mRNA expression in POAG relevant eye tissues and possible changes in disease state. We found that the proteins corresponding to these 65 (candidate) POAG disease genes take part in as few as four common functional molecular networks. Functions attributed to these 4 networks were developmental (dys)function, lipid metabolism, and inflammatory processes. For the 65 POAG disease genes, we reviewed the available (transgenic) mouse models of POAG, which may be useful for future functional studies. Finally, we showed that the 65 (candidate) POAG genes substantially increased the specificity and sensitivity of a discriminative POAG risk test. This suggests that personal risk assessment and personalized medicine for POAG are on the horizon. Taken together, the data presented are essential to comprehend the role of genetic variation in POAG, and may provide leads to understand the pathophysiology of POAG as well as other neurodegenerative disorders, such as Alzheimers disease.

Gene Expression and Functional Annotation of the Human Ciliary Body Epithelia

Purpose The ciliary body (CB) of the human eye consists of the non-pigmented (NPE) and pigmented (PE) neuro-epithelia. We investigated the gene expression of NPE and PE, to shed light on the molecular mechanisms underlying the most important functions of the CB. We also developed molecular signatures for the NPE and PE and studied possible new clues for glaucoma. Methods We isolated NPE and PE cells from seven healthy human donor eyes using laser dissection microscopy. Next, we performed RNA isolation, amplification, labeling and hybridization against 44×k Agilent microarrays. For microarray conformations, we used a literature study, RT-PCRs, and immunohistochemical stainings. We analyzed the gene expression data with R and with the knowledge database Ingenuity. Results The gene expression profiles and functional annotations of the NPE and PE were highly similar. We found that the most important functionalities of the NPE and PE were related to developmental processes, neural nature of the tissue, endocrine and metabolic signaling, and immunological functions. In total 1576 genes differed statistically significantly between NPE and PE. From these genes, at least 3 were cell-specific for the NPE and 143 for the PE. Finally, we observed high expression in the (N)PE of 35 genes previously implicated in molecular mechanisms related to glaucoma. Conclusion Our gene expression analysis suggested that the NPE and PE of the CB were quite similar. Nonetheless, cell-type specific differences were found. The molecular machineries of the human NPE and PE are involved in a range of neuro-endocrinological, developmental and immunological functions, and perhaps glaucoma.

Gene Expression and Functional Annotation of the Human and Mouse Choroid Plexus Epithelium

Background The choroid plexus epithelium (CPE) is a lobed neuro-epithelial structure that forms the outer blood-brain barrier. The CPE protrudes into the brain ventricles and produces the cerebrospinal fluid (CSF), which is crucial for brain homeostasis. Malfunction of the CPE is possibly implicated in disorders like Alzheimer disease, hydrocephalus or glaucoma. To study human genetic diseases and potential new therapies, mouse models are widely used. This requires a detailed knowledge of similarities and differences in gene expression and functional annotation between the species. The aim of this study is to analyze and compare gene expression and functional annotation of healthy human and mouse CPE. Methods We performed 44k Agilent microarray hybridizations with RNA derived from laser dissected healthy human and mouse CPE cells. We functionally annotated and compared the gene expression data of human and mouse CPE using the knowledge database Ingenuity. We searched for common and species specific gene expression patterns and function between human and mouse CPE. We also made a comparison with previously published CPE human and mouse gene expression data. Results Overall, the human and mouse CPE transcriptomes are very similar. Their major functionalities included epithelial junctions, transport, energy production, neuro-endocrine signaling, as well as immunological, neurological and hematological functions and disorders. The mouse CPE presented two additional functions not found in the human CPE: carbohydrate metabolism and a more extensive list of (neural) developmental functions. We found three genes specifically expressed in the mouse CPE compared to human CPE, being ACE, PON1 and TRIM3 and no human specifically expressed CPE genes compared to mouse CPE. Conclusion Human and mouse CPE transcriptomes are very similar, and display many common functionalities. Nonetheless, we also identified a few genes and pathways which suggest that the CPE between mouse and man differ with respect to transport and metabolic functions.

Gene expression and functional annotation of human choroid plexus epithelium failure in Alzheimer's disease

BackgroundAlzheimer’s disease (AD) is the most common form of dementia. AD has a multifactorial disease etiology and is currently untreatable. Multiple genes and molecular mechanisms have been implicated in AD, including ß-amyloid deposition in the brain, neurofibrillary tangle accumulation of hyper-phosphorylated Tau, synaptic failure, oxidative stress and inflammation. Relatively little is known about the role of the blood-brain barriers, especially the blood-cerebrospinal fluid barrier (BCSFB), in AD. The BCSFB is involved in cerebrospinal fluid (CSF) production, maintenance of brain homeostasis and neurodegenerative disorders.ResultsUsing an Agilent platform with common reference design, we performed a large scale gene expression analysis and functional annotation of the Choroid Plexus Epithelium (CPE), which forms the BCSFB. We obtained 2 groups of freshly frozen Choroid Plexus (CP) of 7 human donor brains each, with and without AD: Braak stages (0–1) and (5–6). We cut CP cryo-sections and isolated RNA from cresyl-violet stained, laser dissected CPE cells. Gene expression results were analysed with T-tests (R) and the knowledge-database Ingenuity.We found statistically significantly altered gene expression data sets, biological functions, canonical pathways, molecular networks and functionalities in AD-affected CPE. We observed specific cellular changes due to increased oxidative stress, such as the unfolded protein response, E1F2 and NRF2 signalling and the protein ubiquitin pathway. Most likely, the AD-affected BCSFB barrier becomes more permeable due to downregulation of CLDN5. Finally, our data also predicted down regulation of the glutathione mediated detoxification pathway and the urea cycle in the AD CPE, which suggest that the CPE sink action may be impaired. Remarkably, the expression of a number of genes known to be involved in AD, such as APP, PSEN1, PSEN2, TTR and CLU is moderate to high and remains stable in both healthy and affected CPE. Literature labelling of our new functional molecular networks confirmed multiple previous (molecular) observations in the AD literature and revealed many new ones.ConclusionsWe conclude that CPE failure in AD exists. Combining our data with those of the literature, we propose the following chronological and overlapping chain of events: increased Aß burden on CPE; increased oxidative stress in CPE; despite continuous high expression of TTR: decreased capability of CPE to process amyloid; (pro-) inflammatory and growth factor signalling by CPE; intracellular ubiquitin involvement, remodelling of CPE tight junctions and, finally, cellular atrophy. Our data corroborates the hypothesis that increased BCSFB permeability, especially loss of selective CLDN5-mediated paracellular transport, altered CSF production and CPE sink action, as well as loss of CPE mediated macrophage recruitment contribute to the pathogenesis of AD.

Gene expression-based comparison of the human secretory neuroepithelia of the brain choroid plexus and the ocular ciliary body: potential implications for glaucoma

BackgroundThe neuroepithelia of the choroid plexus (CP) in the brain and the ciliary body (CB) of the eye have common embryological origins and share similar micro-structure and functions. The CP epithelium (CPE) and the non-pigmented epithelium (NPE) of the CB produce the cerebrospinal fluid (CSF) and the aqueous humor (AH) respectively. Production and outflow of the CSF determine the intracranial pressure (ICP); production and outflow of the AH determine the intraocular pressure (IOP). Together, the IOP and ICP determine the translaminar pressure on the optic disc which may be involved in the pathophysiology of primary open angle glaucoma (POAG). The aim of this study was to compare the molecular machinery of the secretory neuroepithelia of the CP and CB (CPE versus NPE) and to determine their potential role in POAG.MethodsWe compared the transcriptomes and functional annotations of healthy human CPE and NPE. Microarray and bioinformatic studies were performed using an Agilent platform and the Ingenuity Knowledge Database (IPA).ResultsBased on gene expression profiles, we found many similar functions for the CPE and NPE including molecular transport, neurological disease processes, and immunological functions. With commonly-used selection criteria (fold-change > 2.5, p-value < 0.05), 14% of the genes were expressed significantly differently between CPE and NPE. When we used stricter selection criteria (fold-change > 5, p-value < 0.001), still 4.5% of the genes were expressed differently, which yielded specific functions for the CPE (ciliary movement and angiogenesis/hematopoiesis) and for the NPE (neurodevelopmental properties). Apart from a few exceptions (e.g. SLC12A2, SLC4A4, SLC4A10, KCNA5, and SCN4B), all ion transport protein coding genes involved in CSF and AH production had similar expression profiles in CPE and NPE. Three POAG disease genes were expressed significantly higher in the CPE than the NPE, namely CDH1, CDKN2B and SIX1.ConclusionsThe transcriptomes of the CPE and NPE were less similar than we previously anticipated. High expression of CSF/AH production genes and candidate POAG disease genes in the CPE and NPE suggest that both might be involved in POAG.

Systematic review of the association between Alzheimer's disease and chronic glaucoma

Dear editor We read with great interest the paper by Tsilis et al entitled “Systematic review of the association between Alzheimer’s disease and chronic glaucoma” published recently in this journal.1 The potential overlap in the pathobiological background of Alzheimer’s disease (AD) and primary open angle glaucoma (POAG) is currently a topic of intense discussion and could provide further insight into both of these complex diseases. Last year, we published an extensive review on POAG and the potential link between AD and POAG.2 Both AD and POAG consist of several clinical subtypes, and are genetically heterogeneous disorders. In our POAG disease model, we found that at least 65 candidate disease genes, together defining several molecular mechanisms (developmental dysfunction, lipid metabolism, and inflammatory processes), underlie the disease.2 For AD, at least 31 genes have been associated with the disease.3 We also showed that, on a molecular level, there is overlap between the molecular mechanisms of POAG and those of AD.4 When comparing two (genetically) heterogeneous disease entities, the conclusion that high heterogeneity and nonassociation will be found seems obvious. The study by Cumurcu et al5 cited by Tsilis et al, concerns a very distinct form of glaucoma, ie, pseudoexfoliation glaucoma, caused uniquely by mutations in the LOXL1 gene,6 and AD. We believe that this study should be considered separately, or pooled with data from other pseudoexfoliation glaucoma (and AD) studies. The data from this study cannot be extrapolated to others. We agree with Tsili et al that large and high-quality association studies, preferably with long follow-up, are needed to clarify the existence and nature of possible associations between POAG and AD. However, at the same time, these studies will only be useful if specific clinical or genetic subtypes of glaucoma and AD are considered. In this respect, we proposed to investigate the existence of a possible association between normal tension glaucoma and AD.2

Type VII Collagen in the Human Accommodation System: Expression in Ciliary Body, Zonules, and Lens Capsule

Purpose To investigate intraocular expression of COL7A1 and its protein product type VII collagen, particularly at the accommodation system. Methods Eyes from 26 human adult donors were used. COL7A1 expression was analyzed in ex vivo ciliary epithelium by microarray. Type VII collagen distribution was examined by Western blot analysis, immunohistochemistry. and immuno-electron microscopy. Results COL7A1 is expressed by pigmented and nonpigmented ciliary epithelia. Type VII collagen is distributed particularly at the strained parts of the accommodation system. Type VII collagen was associated with various basement membranes and with ciliary zonules. Anchoring fibrils were not visualized. Conclusions Type VII collagen distribution at strained areas suggests a supporting role in tissue integrity.

RETINAL THICKNESS CORRELATES WITH PARIETAL CORTICAL ATROPHY ON MRI

CI, 0.73-0.83)], [Ab1-42+NFL] differentiating MCI from ADD [AUROC1⁄40.71 (95% CI, 0.67-0.75)], and t-tau differentiating ADD from FTD [AUROC1⁄40.82 (95% CI, 0.78-0.86). Conclusions: The biomarker combination [Ab1-42+p-tau+NFL] differentiated ADD from HC with good diagnostic accuracy. CSFAb1-42 discriminated MCI from HC with poor diagnostic accuracy. The diagnostic performances of CSF t-tau in distinguishing FTD fromHC and ADD were fair and good, respectively. The combination [Ab1-42+NFL] differentiatedMCI fromADDwith fair diagnostic accuracy. In summary, NFL protein – combined with the core AD biomarkers Ab1-42 and p-tau – is a novel pathophysiological biomarker potentially contributing to improving the diagnostic accuracy in differentiating ADD from HC and, to a lesser degree, ADD from MCI.