Search

Background: Beckwith-Wiedemann syndrome and Silver-Russel syndrome are two imprinting disorders caused by opposite molecular alterations in 11p15.5. With the current diagnostic tests, their molecular diagnosis is challenging due to molecular heterogeneity and mosaic occurrence of the most frequent alterations. As the determination of precise (epi)genotype of patients is relevant as the basis for a personalized treatment, different approaches are needed to increase the sensitivity of diagnostic testing of imprinting disorders., Methods: We established methylation-specific droplet digital PCR approaches (MS-ddPCR) for the two imprinting centers in 11p15.5, and analyzed patients with paternal uniparental disomy of chromosome 11p15.5 (upd(11)pat) and other imprinting defects in the region. The results were compared to those from MS-MLPA (multiplex ligation-dependent probe amplification) and MS-pyrosequencing., Results: MS-ddPCR confirmed the molecular alterations in all patients and the results matched well with MS-MLPA. The results of MS-pyrosequencing varied between different runs, whereas MS-ddPCR results were reproducible., Conclusion: We show for the first time that MS-ddPCR is a reliable and easy applicable method for determination of MS-associated changes in imprinting disorders. It is therefore an additional tool for multimethod diagnostics of imprinting disorders suitable to improve the diagnostic yield., (© 2023 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals LLC.)

Background: Cell-type specific DNA methylation (DNAm) can be employed to determine the numbers of leukocyte subsets in blood. In contrast to conventional methods for leukocyte counts, which are based on cellular morphology or surface marker protein expression, the cellular deconvolution based on DNAm levels is applicable for frozen or dried blood. Here, we further enhanced targeted DNAm assays for leukocyte counts in clinical application., Methods: DNAm profiles of 40 different studies were compiled to identify CG dinucleotides (CpGs) with cell-type specific DNAm using a computational framework, CimpleG. DNAm levels at these CpGs were then measured with digital droplet PCR in venous blood from 160 healthy donors and 150 patients with various hematological disorders. Deconvolution was further validated with venous blood (n = 75) and capillary blood (n = 31) that was dried on Whatman paper or on Mitra microsampling devices., Results: In venous blood, automated cell counting or flow cytometry correlated well with epigenetic estimates of relative leukocyte counts for granulocytes (r = 0.95), lymphocytes (r = 0.97), monocytes (r = 0.82), CD4 T cells (r = 0.84), CD8 T cells (r = 0.94), B cells (r = 0.96), and NK cells (r = 0.72). Similar correlations and precisions were achieved for dried blood samples. Spike-in with a reference plasmid enabled accurate epigenetic estimation of absolute leukocyte counts from dried blood samples, correlating with conventional venous (r = 0.86) and capillary (r = 0.80) blood measurements., Conclusions: The advanced selection of cell-type specific CpGs and utilization of digital droplet PCR analysis provided accurate epigenetic blood counts. Analysis of dried blood facilitates self-sampling with a finger prick, thereby enabling easier accessibility to testing., (© Association for Diagnostics & Laboratory Medicine 2023. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Differential leukocyte counts are usually measured based on cellular morphology or surface marker expression. It has recently been shown that leukocyte counts can also be determined by cell-type-specific DNA methylation (DNAm). Such epigenetic leukocyte counting is applicable to small blood volumes and even frozen material, but for clinical translation, the method needs to be further refined and validated., Methods: We further optimized and validated targeted DNAm assays for leukocyte deconvolution using 332 venous and 122 capillary blood samples from healthy donors. In addition, we tested 36 samples from ring trials and venous blood from 266 patients diagnosed with different hematological diseases. Deconvolution of cell types was determined with various models using DNAm values obtained by pyrosequencing or digital droplet PCR (ddPCR)., Results: Relative leukocyte quantification correlated with conventional blood counts for granulocytes, lymphocytes, B cells, T cells (CD4 or CD8), natural killer cells, and monocytes with pyrosequencing (r = 0.84; r = 0.82; r = 0.58; r = 0.50; r = 0.70; r = 0.61; and r = 0.59, respectively) and ddPCR measurements (r = 0.65; r = 0.79; r = 0.56; r = 0.57; r = 0.75; r = 0.49; and r = 0.46, respectively). In some patients, particularly with hematopoietic malignancies, we observed outliers in epigenetic leukocyte counts, which could be discerned if relative proportions of leukocyte subsets did not sum up to 100%. Furthermore, absolute quantification was obtained by spiking blood samples with a reference plasmid of known copy number., Conclusions: Targeted DNAm analysis by pyrosequencing or ddPCR is a valid alternative to quantify leukocyte subsets, but some assays require further optimization., (© American Association for Clinical Chemistry 2022.)

Purpose: The trabecular meshwork (TM) is situated in the most frontal part of the eye and is thought to play an important role in the regulation of the eye pressure. However, this tissue is rather difficult to harvest for research. The purpose of this study is therefore to integrate the existing gene expression data of the healthy TM to increase sample size and identify its signature genes and pathways. This provides a robust reference for the study of molecular disease processes and supports the selection of candidate target genes for new treatments., Methods: A systematic search identified microarray data of healthy TM tissue. After quality control, datasets of low quality and deviating samples were excluded. Remaining individuals were jointly normalized and integrated into one database. The average gene expression of each tested gene over all individuals was calculated. The 25% genes with the highest average expression were identified as the most active genes in the healthy TM and used as input for pathway and network analysis. Additionally, ubiquitous pathways and genes were identified and excluded from the results. Lastly, we identified genes which are likely to be TM-specific., Results: The gene expression data of 44 individuals, obtained from 18 datasets, were jointly normalized. Ubiquitous genes (n = 688) and ubiquitous pathways (n = 73) were identified and excluded. Following, 1882 genes and 211 pathways were identified as the signature genes and pathways of the healthy TM. Pathway analysis revealed multiple molecular processes of which some were already known to be active in the TM, for example extracellular matrix and elastic fiber formation. Forty-six candidate TM-specific genes were identified. These consist mainly of pseudogenes or novel transcripts of which the function is unknown., Conclusions: In this comprehensive meta-analysis we identified non-ubiquitous genes and pathways that form the signature of the functioning of the healthy TM. Additionally, 46 candidate TM-specific genes were identified. This method can also be used for other tissues that are difficult to obtain for study., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Although mitochondrial dysfunction is the known cause of primary mitochondrial disease, mitochondrial dysfunction is often difficult to measure and prove, especially when biopsies of affected tissue are not available. In order to identify blood biomarkers of mitochondrial dysfunction, we reviewed studies that measured blood biomarkers in genetically, clinically or biochemically confirmed primary mitochondrial disease patients. In this way, we were certain that there was an underlying mitochondrial dysfunction which could validate the biomarker. We found biomarkers of three classes: 1) functional markers measured in blood cells, 2) biochemical markers of serum/plasma and 3) DNA markers. While none of the reviewed single biomarkers may perfectly reveal all underlying mitochondrial dysfunction, combining biomarkers that cover different aspects of mitochondrial impairment probably is a good strategy. This biomarker panel may assist in the diagnosis of primary mitochondrial disease patients. As mitochondrial dysfunction may also play a significant role in the pathophysiology of multifactorial disorders such as Alzheimer's disease and glaucoma, the panel may serve to assess mitochondrial dysfunction in complex multifactorial diseases as well and enable selection of patients who could benefit from therapies targeting mitochondria., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Purpose: Identify differentially expressed microRNAs (miRNAs) in aqueous humor (AH) and blood of primary open-angle glaucoma (POAG) patients by using small RNA sequencing. These may provide insight into POAG pathophysiology or serve as diagnostic biomarker., Methods: AH and plasma of nine POAG patients and 10 cataract control patients were small RNA sequenced on Illumina NovaSeq 6000. Identification of gene transcripts targeted by differentially expressed miRNAs was done with miRWalk and MirPath. These targets were used for pathway analysis and Gene Ontology enrichment. Diagnostic potential was evaluated by receiver operating characteristics analysis., Results: We identified 715 miRNAs in plasma and 62 miRNAs in AH. Plasma miRNA profile did not differ between POAG and control. In contrast, in AH, seven miRNAs were differentially expressed. Hsa-miR-30a-3p, hsa-miR-143-3p, hsa-miR-211-5p, and hsa-miR-221-3p were upregulated, whereas hsa-miR-92a-3p, hsa-miR-451a, and hsa-miR-486-5p were downregulated in POAG. Compared to previous studies, hsa-mir-143-3p, hsa-miR-211-5p, and hsa-miR-221-3p were reported previously, strengthening their involvement in POAG whereas hsa-miR-30a-3p, hsa-miR-92a-3p, and hsa-miR-486-5p are implicated in POAG for the first time. Identified gene transcripts were involved in several pathways, some implicated in glaucoma before (e.g., TGF-β and neurotrophin signaling), whereas others are new (e.g., prolactin and apelin signaling). In respect to diagnostics, AH concentration of hsa-mir-143-3p had an area under the curve (AUC) of 0.889. Combined with hsa-miR-221-3p, AUC improved to 0.96., Conclusions: Small RNA sequencing identified seven differentially expressed miRNAs in AH of POAG patients. The differentially expressed miRNAs may be useful as POAG biomarkers or could become targets for new therapeutic strategies.

Aim: Recently, the level of growth differentiation factor 15 (GDF-15) in blood, was proposed as biomarker to detect mitochondrial dysfunction. In the current study, we evaluate this biomarker in open-angle glaucoma (OAG), as there is increasing evidence that mitochondrial dysfunction plays a role in the pathophysiology of this disease., Methods: Plasma GDF-15 concentrations were measured with ELISA in 200 OAG patients and 61 age-matched controls (cataract without glaucoma). The OAG patient group consisted of high tension glaucoma (HTG; n = 162) and normal tension glaucoma (NTG; n = 38). Groups were compared using the Kruskal-Wallis nonparametric test with Dunn's multiple comparison post-hoc correction. GDF-15 concentration was corrected for confounders identified with forward linear regression models., Results: Before correcting for confounders, median plasma GDF-15 levels was significantly lower in the combined OAG group (p = 0.04), but not when analysing HTG and NTG patients separately. Forward linear regression analysis showed that age, gender, smoking and systemic hypertension were significant confounders affecting GDF-15 levels. After correction for these confounders, GDF-15 levels in OAG patients were no longer significantly different from controls. Subgroup analysis of the glaucoma patients did not show a correlation between disease severity and plasma GDF-15, but did reveal that for NTG patients, intake of dietary supplements, which potentially improve mitochondrial function, correlated with lower plasma GDF-15., Conclusion: The present study suggests that plasma GDF-15 is not suited as biomarker of mitochondrial dysfunction in OAG patients., Competing Interests: The authors have declared that no competing interests exist.

Introduction: We recently performed a combined analysis of publicly available proteomic studies of aqueous humor (AH) of patients with primary open angle glaucoma (POAG). This analysis revealed changes in complement protein concentrations in the AH of progressive POAG patients, which suggested that the complement system may play a role in POAG progression. As the proteomic studies could not provide information on the activity of the complement system, we addressed this question in the current study., Methods: Blood serum and AH were obtained from 30 patients: 10 progressive POAG, 10 stable POAG and, as controls, 10 cataract patients. Glaucoma patients with a visual field Mean Deviation (MD) change of at least 1.0 dB/year were considered progressive; a MD change of less than 0.5 dB/year was considered stable. The ratio between the levels of complement factors C3a and C3 was used as indicator for activation of the complement cascade. The factors were measured with commercially available ELISA kits., Results: AH levels of complement factors C3 and C3a did not significantly differ between groups. In serum, complement factor C3 did not differ between groups whereas C3a was significantly elevated in progressive POAG patients compared to controls (p < 0.05). The resulting complement C3a/C3 ratio was significantly higher in progressive POAG patients in both AH (p < 0.05) and serum (p < 0.01), and this ratio significantly correlated between the two body fluids (p < 0.001). Furthermore, there was a strong correlation between disease progression and C3a/C3 activation ratio both in AH (p < 0.01) and in serum (p < 0.001). The higher the complement C3a/C3 ratio, the faster the disease progression., Conclusion: Significant increases in AH and serum complement C3a/C3 ratios were observed in progressive POAG patients but not in stable POAG patients. Furthermore, the complement C3a/C3 ratio correlated strongly with the rate of disease progression in both AH and serum. These findings suggest that activation of the complement system plays a role in glaucoma progression and that progressive glaucoma patients may have systemic changes in complement activation., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Analysis of the proteins of the aqueous humor can help to elucidate the complex pathogenesis of primary open angle glaucoma. Thanks to advances in liquid chromatography tandem mass spectrometry (LC-MS/MS) it is now possible to identify hundreds of proteins in individual aqueous humor samples without the need to pool samples. We performed a systematic literature search to find publications that performed LC-MS/MS on aqueous humor samples of glaucoma patients and of non-glaucomatous controls. Of the seven publications that we found, we obtained the raw data of three publications. These three studies used glaucoma patients that were clinically similar (i.e. undergoing glaucoma filtration surgery) which prompted us to reanalyse and combine their data. Raw data of each study were analysed separately with the latest version of MaxQuant (version v1.6.11.0). Outcome files were exported to Microsoft Excel. Samples belonging to the same patient were averaged to obtain peptide expression values per individual. We compared the overlap of identified proteins using the VLOOKUP function of Excel and a publicly available Venn diagram software. For the peptide sequences that can belong to multiple proteins (usually of the same protein family), we initially included all possibly identified proteins. This ensured that we would not miss a potential overlap between the studies due to differences in identified peptide counts. Next, of those peptides of which we compared multiple proteins, only one unique protein was included in our analysis i.e. either the protein overlapping between studies or in case of no overlap, the protein that had the highest identified peptide count. This yielded 639 unique proteins detected in aqueous humor of either glaucoma patients or non-glaucomatous controls. In our manuscript entitled "The aqueous humor proteome of primary open angle glaucoma: An extensive review" [1], we further analysed this dataset. The dataset was exported to Perseus (version 1.6.5.0). We removed contaminants and filtered for proteins detected with high confidence, i.e. in more than 70% of the samples of at least one study. This yielded 248 proteins of which we compared the expression in glaucoma patients against control patients. Gene ontology enrichment analysis and pathway analysis was used to interpret the results. The unfiltered dataset reported in this data article and the approach reported here to reanalyse and combine raw data of different studies can be applied by other glaucoma researchers to gain more insight in the pathogenesis of glaucoma., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships which have, or could be perceived to have, influenced the work reported in this article., (© 2020 The Author(s).)

Background: We reviewed the literature on the aqueous humor (AH) proteome of primary open angle glaucoma (POAG) patients in order to obtain deeper insight into the pathophysiology of POAG., Methods: We searched Pubmed and Embase up to May 2019 for studies that compared AH protein composition between POAG (cases) and cataract (controls). Untargeted studies (measuring the whole proteome, by LC-MS/MS) were divided into two subgroups depending on the type of surgery during which POAG AH was collected: glaucoma filtration surgery (subgroup 1) or cataract surgery (subgroup 2). We reanalyzed the raw data (subgroup 1) or combined the reported data (subgroup 2) to perform GO enrichment (GOrilla) and pathway analysis (Pathvisio)., Results: Out of 93 eligible proteomic studies, seven were untargeted studies that identified 863 AH proteins. We observed 73 differentially expressed proteins in subgroup 1 and 87 differentially expressed proteins in subgroup 2. Both subgroups were characterized by activation of the acute immune response, dysregulation of folate metabolism and dysregulation of the selenium micronutrient network. For subgroup 1 but not for subgroup 2, proteins of the complement system were significantly enriched., Conclusion: AH proteome of POAG patients shows strong activation of the immune system. In addition, analysis suggests dysregulation of folate metabolism and dysregulation of selenium as underlying contributors. In view of their glaucoma surgery, POAG patients of subgroup 1 most likely are progressive whereas POAG patients in subgroup 2 most likely have stable POAG. The proteome difference between these subgroups suggests that the complement system plays a role in POAG progression., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Purpose/Aim: Many genes have been associated with primary open-angle glaucoma (POAG). Knowing exactly where they are expressed in the eye helps to unravel POAG pathology and to select optimal targets for intervention. We investigated whether RNA in situ hybridization (RNA-ISH) is a convenient technique to obtain detailed pan-ocular expression data of these genes. We tested this for four diverse candidate POAG genes, selected because of unclear ocular distribution ( F5 and Dusp1) and relevance for potential new therapies ( Tnf, Tgfβr3 ). Optn , a POAG gene with well-known ocular expression pattern served as control. Methods: We made a list of candidate glaucoma genes reported in genetic studies. A table of their ocular expression at the tissue level was compiled using publicly available microarray data (the ocular tissue database). To add cellular detail we performed RNA-ISH for Optn, Tnf, Tgfβr3, F5 , and Dusp1 on eyes of healthy, 2-month-old, pigmented, and albino mice. Results: Expression of the Optn control matched with published immunohistochemistry data. Ocular expression of Tnf was generally low, with patches of higher Tnf expression, superficially in the corneal epithelium. F5 had a restricted expression pattern with high expression in the nonpigmented ciliary body epithelium and moderate expression in the peripapillary region. Tgfβr3 and Dusp1 showed ubiquitous expression. Conclusions: RNA-ISH is a suitable technique to determine the ocular expression pattern of POAG genes, adding meaningful cellular detail to existing microarray expression data. For instance, the high expression of F5 in the nonpigmented ciliary body epithelium suggests a role of this gene in aqueous humor dynamics and intraocular pressure. In addition, the ubiquitous expression of Tgfβr3 has implications for designing TGF-β-related glaucoma therapies, with respect to side effects. Creating pan-ocular expression maps of POAG genes with RNA-ISH will help to identify POAG pathways in specific cell types and to select targets for drug development.