Dysregulation of the alternative pathway (AP) of complement cascade has been implicated in the pathogenesis of age-related macular degeneration (AMD), the leading cause of blindness in the elderly. ...To further test the hypothesis that defective control of complement activation underlies AMD, parameters of complement activation in blood plasma were determined together with disease-associated genetic markers in AMD patients. Plasma concentrations of activation products C3d, Ba, C3a, C5a, SC5b-9, substrate proteins C3, C4, factor B and regulators factor H and factor D were quantified in patients (n = 112) and controls (n = 67). Subjects were analyzed for single nucleotide polymorphisms in factor H (CFH), factor B-C2 (BF-C2) and complement C3 (C3) genes which were previously found to be associated with AMD. All activation products, especially markers of chronic complement activation Ba and C3d (p<0.001), were significantly elevated in AMD patients compared to controls. Similar alterations were observed in factor D, but not in C3, C4 or factor H. Logistic regression analysis revealed better discriminative accuracy of a model that is based only on complement activation markers Ba, C3d and factor D compared to a model based on genetic markers of the complement system within our study population. In both the controls' and AMD patients' group, the protein markers of complement activation were correlated with CFH haplotypes.This study is the first to show systemic complement activation in AMD patients. This suggests that AMD is a systemic disease with local disease manifestation at the ageing macula. Furthermore, the data provide evidence for an association of systemic activation of the alternative complement pathway with genetic variants of CFH that were previously linked to AMD susceptibility.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
There is no international consensus up to which age women with a diagnosis of triple-negative breast cancer (TNBC) and no family history of breast or ovarian cancer should be offered genetic testing ...for germline BRCA1 and BRCA2 (gBRCA) mutations. Here, we explored the association of age at TNBC diagnosis with the prevalence of pathogenic gBRCA mutations in this patient group.
The study comprised 802 women (median age 40 years, range 19-76) with oestrogen receptor, progesterone receptor, and human epidermal growth factor receptor type 2 negative breast cancers, who had no relatives with breast or ovarian cancer. All women were tested for pathogenic gBRCA mutations. Logistic regression analysis was used to explore the association between age at TNBC diagnosis and the presence of a pathogenic gBRCA mutation.
A total of 127 women with TNBC (15.8%) were gBRCA mutation carriers (BRCA1: n = 118, 14.7%; BRCA2: n = 9, 1.1%). The mutation prevalence was 32.9% in the age group 20-29 years compared to 6.9% in the age group 60-69 years. Logistic regression analysis revealed a significant increase of mutation frequency with decreasing age at diagnosis (odds ratio 1.87 per 10 year decrease, 95%CI 1.50-2.32, p < 0.001). gBRCA mutation risk was predicted to be > 10% for women diagnosed below approximately 50 years.
Based on the general understanding that a heterozygous mutation probability of 10% or greater justifies gBRCA mutation screening, women with TNBC diagnosed before the age of 50 years and no familial history of breast and ovarian cancer should be tested for gBRCA mutations. In Germany, this would concern approximately 880 women with newly diagnosed TNBC per year, of whom approximately 150 are expected to be identified as carriers of a pathogenic gBRCA mutation.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Age-related macular degeneration (AMD) is a common threat to vision. While classification of disease stages is critical to understanding disease risk and progression, several systems based on color ...fundus photographs are known. Most of these require in-depth and time-consuming analysis of fundus images. Herein, we present an automated computer-based classification algorithm.
Algorithm development for AMD classification based on a large collection of color fundus images. Validation is performed on a cross-sectional, population-based study.
We included 120 656 manually graded color fundus images from 3654 Age-Related Eye Disease Study (AREDS) participants. AREDS participants were >55 years of age, and non-AMD sight-threatening diseases were excluded at recruitment. In addition, performance of our algorithm was evaluated in 5555 fundus images from the population-based Kooperative Gesundheitsforschung in der Region Augsburg (KORA; Cooperative Health Research in the Region of Augsburg) study.
We defined 13 classes (9 AREDS steps, 3 late AMD stages, and 1 for ungradable images) and trained several convolution deep learning architectures. An ensemble of network architectures improved prediction accuracy. An independent dataset was used to evaluate the performance of our algorithm in a population-based study.
κ Statistics and accuracy to evaluate the concordance between predicted and expert human grader classification.
A network ensemble of 6 different neural net architectures predicted the 13 classes in the AREDS test set with a quadratic weighted κ of 92% (95% confidence interval, 89%–92%) and an overall accuracy of 63.3%. In the independent KORA dataset, images wrongly classified as AMD were mainly the result of a macular reflex observed in young individuals. By restricting the KORA analysis to individuals >55 years of age and prior exclusion of other retinopathies, the weighted and unweighted κ increased to 50% and 63%, respectively. Importantly, the algorithm detected 84.2% of all fundus images with definite signs of early or late AMD. Overall, 94.3% of healthy fundus images were classified correctly.
Our deep learning algoritm revealed a weighted κ outperforming human graders in the AREDS study and is suitable to classify AMD fundus images in other datasets using individuals >55 years of age.
X-linked juvenile retinoschisis (XLRS, MIM 312700) is a common early onset macular degeneration in males characterized by mild to severe loss in visual acuity, splitting of retinal layers, and a ...reduction in the b-wave of the electroretinogram (ERG). The RS1 gene (MIM 300839) associated with the disease encodes retinoschisin, a 224 amino acid protein containing a discoidin domain as the major structural unit, an N-terminal cleavable signal sequence, and regions responsible for subunit oligomerization. Retinoschisin is secreted from retinal cells as a disulphide-linked homo-octameric complex which binds to the surface of photoreceptors and bipolar cells to help maintain the integrity of the retina. Over 190 disease-causing mutations in the RS1 gene are known with most mutations occurring as non-synonymous changes in the discoidin domain. Cell expression studies have shown that disease-associated missense mutations in the discoidin domain cause severe protein misfolding and retention in the endoplasmic reticulum, mutations in the signal sequence result in aberrant protein synthesis, and mutations in regions flanking the discoidin domain cause defective disulphide-linked subunit assembly, all of which produce a non-functional protein. Knockout mice deficient in retinoschisin have been generated and shown to display most of the characteristic features found in XLRS patients. Recombinant adeno-associated virus (rAAV) mediated delivery of the normal RS1 gene to the retina of young knockout mice result in long-term retinoschisin expression and rescue of retinal structure and function providing a ‘proof of concept’ that gene therapy may be an effective treatment for XLRS.
Sphingolipids are bioactive molecules associated with oxidative stress, inflammation, and neurodegenerative diseases, but poorly studied in the context of age-related macular degeneration (AMD), a ...prevalent sight-threatening disease of the ageing retina. Here, we found higher serum levels of hexosylceramide (HexCer) d18:1/16:0 in patients with choroidal neovascularization (CNV) and geographic atrophy (GA), two manifestations of late stage AMD, and higher ceramide (Cer) d18:1/16:0 levels in GA patients. A sensitivity analysis of genetic variants known to be associated with late stage AMD showed that rs1061170 (p.Y402H) in the complement factor H (CFH) gene influences the association of Cer d18:1/16:0 with GA. To understand the possible influence of this genetic variant on ceramide levels, we established a cell-based assay to test the modulation of genes in the ceramide metabolism by factor H-like protein 1 (FHL-1), an alternative splicing variant of CFH that also harbors the 402 residue. We first showed that malondialdehyde-acetaldehyde adducts, an oxidation product commonly found in AMD retinas, induces an increase in ceramide levels in WERI-Rb1 cells in accordance with an increased expression of ceramide synthesis genes. Then, we observed that cells exposed to the non-risk FHL-1:Y402, but not the risk associated variant FHL-1:H402 or full-length CFH, downregulated ceramide synthase 2 and ceramide glucosyltransferase gene expression. Together, our findings show that serum ceramide and hexosylceramide species are altered in AMD patients and that ceramide levels may be influenced by AMD associated risk variants.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The extent of aneuploidy of the sex chromosomes increases with age in human leukocytes. Here, we re-explore the dynamics of normal loss of the Y chromosome (
LOY
) with age based on microarray data ...using two exponential models and two different ways to estimate the fraction of
LOY
. This analysis shows the existence of a significant correlation between the fraction of
LOY
estimated from molecular cytogenetics and genotyping microarray data. Although the specific estimates of the parameters for the two exponential models are different from those derived from cytogenetics data, the present analysis in an independent dataset of normal individuals confirms that X0 cells have a selective advantage over XY cells. Moreover, patients with age-related macular degeneration display higher fraction of
LOY
values and seem to have a predisposition to lose their Y chromosome even at young ages compared to control individuals. As there are no data available for the same individuals at different time points, the parameters reported here are average values drawn from population analyses.
Significant association signals from genome-wide association studies (GWAS) point to genomic regions of interest. However, for most loci the causative genetic variant remains undefined. Determining ...expression quantitative trait loci (eQTL) in a disease relevant tissue is an excellent approach to zoom in on disease- or trait-associated association signals and hitherto on relevant disease mechanisms. To this end, we explored regulation of gene expression in healthy retina (n = 311) and generated the largest cis-eQTL data set available to date. Genotype- and RNA-Seq data underwent rigorous quality control protocols before FastQTL was applied to assess the influence of genetic markers on local (cis) gene expression. Our analysis identified 403,151 significant eQTL variants (eVariants) that regulate 3,007 genes (eGenes) (Q-Value < 0.05). A conditional analysis revealed 744 independent secondary eQTL signals for 598 of the 3,007 eGenes. Interestingly, 99,165 (24.71%) of all unique eVariants regulate the expression of more than one eGene. Filtering the dataset for eVariants regulating three or more eGenes revealed 96 potential regulatory clusters. Of these, 31 harbour 130 genes which are partially regulated by the same genetic signal. To correlate eQTL and association signals, GWAS data from twelve complex eye diseases or traits were included and resulted in identification of 80 eGenes with potential association. Remarkably, expression of 10 genes is regulated by eVariants associated with multiple eye diseases or traits. In conclusion, we generated a unique catalogue of gene expression regulation in healthy retinal tissue and applied this resource to identify potentially pleiotropic effects in highly prevalent human eye diseases. Our study provides an excellent basis to further explore mechanisms of various retinal disease etiologies.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
A frequent deletion of complement factor H (CFH)-related genes CFHR3 and CFHR1 (ΔCFHR3/CFHR1) is considered to have a protective effect against age-related macular degeneration (AMD), although the ...underlying mechanism remains elusive. The deletion seems to be linked to one of the two protective CFH haplotypes which are both tagged by the protective allele of single nucleotide polymorphism rs2274700 (CFH:A473A). In a German cohort of 530 AMD patients, we now show that protection against AMD conferred by ΔCFHR3/CFHR1 is independent of the effects of rs2274700 and rs1061170 (CFH:Y402H). This suggests a functional role of CFHR1 and/or CFHR3 in disease pathogenesis. We therefore characterized the CFHR3 function and identified CFHR3 as a novel human complement regulator that inhibits C3 convertase activity. CFHR3 displays anti-inflammatory effects by blocking C5a generation and C5a-mediated chemoattraction of neutrophils. In addition, CFHR3 and CFHR1 compete with factor H for binding to the central complement component C3. Thus, deficiency of CFHR3 and CFHR1 results in a loss of complement control but enhances local regulation by factor H. Our findings allude to a critical balance between the complement regulators CFHR3, CFHR1 and factor H and further emphasize the central role of complement regulation in AMD pathology.
MicroRNAs (miRNAs) regulate pathways involved in cell differentiation, proliferation, development, and apoptosis by degradation of target mRNAs and/or repression of their translation. Although the ...single nucleotide polymorphisms (SNPs) in miRNAs target sites have been studied, the effects of SNPs in miRNAs are largely unknown. In our study, we first systematically sequenced miRNA genes reported to be involved in breast cancer to identify/verify SNPs. We analyzed four SNPs, one located in the pre-miRNA and the other three located in miRNA flanking regions, for a putative association with breast cancer risk. The SNP rs895819, located in the terminal loop of pre-miRNA-27a, showed a protective effect. In a large familial breast cancer study cohort, the rare G allele of rs895819 was found to be less frequent in the cases than in the controls, indicating a reduced familial breast cancer risk (G vs. A: OR = 0.88, 95% CI 0.78-0.99, P = 0.0287). Furthermore, age stratification revealed that the protective effect was mainly observed in the age group < 50 years of age (G vs. A: OR = 0.83, 95% CI 0.70-0.98, P = 0.0314), whereas no significant effect was observed in the age group ≥ 50 years of age, indicating a possible hormone-related effect. It has been shown that artificial mutations in the terminal loop of miR-27a can block the maturation process of the miRNA. We hypothesize that the G-variant of rs895819 might impair the maturation of the oncogenic miR-27a and thus, is associated with familial breast cancer risk.
Ligand-driven modulation of the mitochondrial translocator protein 18 kDa (TSPO) was recently described to dampen the neuroinflammatory response of microglia in a retinal light damage model resulting ...in protective effects on photoreceptors. We characterized the effects of the TSPO ligand XBD173 in the postischemic retina focusing on changes in the response pattern of the major glial cell types of the retina-microglia and Müller cells.
Retinal ischemia was induced by increasing the intraocular pressure for 60 min followed by reperfusion of the tissue in mice. On retinal cell types enriched via immunomagnetic separation expression analysis of TSPO, its ligand diazepam-binding inhibitor (DBI) and markers of glial activation were performed at transcript and protein level using RNA sequencing, qRT-PCR, lipid chromatography-mass spectrometry, and immunofluorescent labeling. Data on cell morphology and numbers were assessed in retinal slice and flatmount preparations. The retinal functional integrity was determined by electroretinogram recordings.
We demonstrate that TSPO is expressed by Müller cells, microglia, vascular cells, retinal pigment epithelium (RPE) of the healthy and postischemic retina, but only at low levels in retinal neurons. While an alleviated neurodegeneration upon XBD173 treatment was found in postischemic retinae as compared to vehicle controls, this neuroprotective effect of XBD173 is mediated putatively by its action on retinal glia. After transient ischemia, TSPO as a marker of activation was upregulated to similar levels in microglia as compared to their counterparts in healthy retinae irrespective of the treatment regimen. However, less microglia were found in XBD173-treated postischemic retinae at 3 days post-surgery (dps) which displayed a more ramified morphology than in retinae of vehicle-treated mice indicating a dampened microglia activation. Müller cells, the major retinal macroglia, show upregulation of the typical gliosis marker GFAP. Importantly, glutamine synthetase was more stably expressed in Müller glia of XBD173-treated postischemic retinae and homeostatic functions such as cellular volume regulation typically diminished in gliotic Müller cells remained functional.
In sum, our data imply that beneficial effects of XBD173 treatment on the postischemic survival of inner retinal neurons were primarily mediated by stabilizing neurosupportive functions of glial cells.
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