Epigenome-metabolism nexus in the retina: implications for aging and disease.

Intimate links between epigenome modifications and metabolites allude to a crucial role of cellular metabolism in transcriptional regulation. Retina, being a highly metabolic tissue, adapts by integrating inputs from genetic, epigenetic, and extracellular signals. Precise global epigenomic signatures guide development and homeostasis of the intricate retinal structure and function. Epigenomic and metabolic realignment are hallmarks of aging and highlight a link of the epigenome-metabolism nexus with aging-associated multifactorial traits affecting the retina, including age-related macular degeneration and glaucoma. Here, we focus on emerging principles of epigenomic and metabolic control of retinal gene regulation, with emphasis on their contribution to human disease. In addition, we discuss potential mitigation strategies involving lifestyle changes that target the epigenome-metabolome relationship for maintaining retinal function.

Whole genome sequencing of 4,787 individuals identifies gene-based rare variants in age-related macular degeneration.

Genome-wide association studies have contributed extensively to the discovery of disease-associated common variants. However, the genetic contribution to complex traits is still largely difficult to interpret. We report a genome-wide association study of 2394 cases and 2393 controls for age-related macular degeneration (AMD) via whole-genome sequencing, with 46.9 million genetic variants. Our study reveals significant single-variant association signals at four loci and independent gene-based signals in CFH, C2, C3, and NRTN. Using data from the Exome Aggregation Consortium (ExAC) for a gene-based test, we demonstrate an enrichment of predicted rare loss-of-function variants in CFH, CFI, and an as-yet unreported gene in AMD, ORMDL2. Our method of using a large variant list without individual-level genotypes as an external reference provides a flexible and convenient approach to leverage the publicly available variant datasets to augment the search for rare variant associations, which can explain additional disease risk in AMD.

GATD3A, a mitochondrial deglycase with evolutionary origins from gammaproteobacteria, restricts the formation of advanced glycation end products.

BACKGROUND: Functional complexity of the eukaryotic mitochondrial proteome is augmented by independent gene acquisition from bacteria since its endosymbiotic origins. Mammalian homologs of many ancestral mitochondrial proteins have uncharacterized catalytic activities. Recent forward genetic approaches attributed functions to proteins in established metabolic pathways, thereby limiting the possibility of identifying novel biology relevant to human disease. We undertook a bottom-up biochemistry approach to discern evolutionarily conserved mitochondrial proteins with catalytic potential. RESULTS: Here, we identify a Parkinson-associated DJ-1/PARK7-like protein-glutamine amidotransferase-like class 1 domain-containing 3A (GATD3A), with bacterial evolutionary affinities although not from alphaproteobacteria. We demonstrate that GATD3A localizes to the mitochondrial matrix and functions as a deglycase. Through its amidolysis domain, GATD3A removes non-enzymatic chemical modifications produced during the Maillard reaction between dicarbonyls and amines of nucleotides and amino acids. GATD3A interacts with factors involved in mitochondrial mRNA processing and translation, suggestive of a role in maintaining integrity of important biomolecules through its deglycase activity. The loss of GATD3A in mice is associated with accumulation of advanced glycation end products (AGEs) and altered mitochondrial dynamics. CONCLUSIONS: An evolutionary perspective helped us prioritize a previously uncharacterized but predicted mitochondrial protein GATD3A, which mediates the removal of early glycation intermediates. GATD3A restricts the formation of AGEs in mitochondria and is a relevant target for diseases where AGE deposition is a pathological hallmark.

Signaling alterations in oral keratinocytes in response to shisha and crude tobacco extract.

BACKGROUND: Tobacco consumption in smoking and non-smoking forms has been consequential in the rise of oral cancer cases. Among different forms, epidemiological studies from Middle Eastern countries and rural parts of northern India have reported increasing association of oral cancer with waterpipe (hookah) smoking. However, molecular mechanisms and role played by waterpipe smoking in the onset of oral carcinogenesis remains unexplored. METHODS: In this study, immortalized normal human oral keratinocytes were chronically treated with extracts of two varieties of waterpipe tobacco-crude tobacco and processed shisha. Phenotypic changes and molecular aberrations were examined using cell culture-based assays and mass spectrometry-based quantitative proteomic analysis, respectively. Bioinformatics analysis was utilized to analyze proteomics data and identify dysregulated pathways. RESULTS: Our data indicate that chronic treatment with waterpipe tobacco extracts increased proliferation, invasion, migration, and significant dysregulation of protein expression in oral keratinocytes. Altered expression of proteins involved in interferon signaling pathway were observed with both varieties of tobacco. Overexpression of cholesterol metabolism and vesicle-mediated transport proteins were identified exclusively in cells treated with crude tobacco extract. Bioinformatics analyses revealed different oncogenic response in oral cells based on the type of waterpipe tobacco used. CONCLUSIONS: This study may serve as a useful resource in understanding the early onset of oral cancer attributed to waterpipe smoking.

Integrating transcriptomic and proteomic data for accurate assembly and annotation of genomes.

Complementing genome sequence with deep transcriptome and proteome data could enable more accurate assembly and annotation of newly sequenced genomes. Here, we provide a proof-of-concept of an integrated approach for analysis of the genome and proteome of Anopheles stephensi, which is one of the most important vectors of the malaria parasite. To achieve broad coverage of genes, we carried out transcriptome sequencing and deep proteome profiling of multiple anatomically distinct sites. Based on transcriptomic data alone, we identified and corrected 535 events of incomplete genome assembly involving 1196 scaffolds and 868 protein-coding gene models. This proteogenomic approach enabled us to add 365 genes that were missed during genome annotation and identify 917 gene correction events through discovery of 151 novel exons, 297 protein extensions, 231 exon extensions, 192 novel protein start sites, 19 novel translational frames, 28 events of joining of exons, and 76 events of joining of adjacent genes as a single gene. Incorporation of proteomic evidence allowed us to change the designation of more than 87 predicted "noncoding RNAs" to conventional mRNAs coded by protein-coding genes. Importantly, extension of the newly corrected genome assemblies and gene models to 15 other newly assembled Anopheline genomes led to the discovery of a large number of apparent discrepancies in assembly and annotation of these genomes. Our data provide a framework for how future genome sequencing efforts should incorporate transcriptomic and proteomic analysis in combination with simultaneous manual curation to achieve near complete assembly and accurate annotation of genomes.

Phosphoproteomic analysis identifies CLK1 as a novel therapeutic target in gastric cancer.

BACKGROUND: Phosphorylation is an important regulatory mechanism of protein activity in cells. Studies in various cancers have reported perturbations in kinases resulting in aberrant phosphorylation of oncoproteins and tumor suppressor proteins. METHODS: In this study, we carried out quantitative phosphoproteomic analysis of gastric cancer tissues and corresponding xenograft samples. Using these data, we employed bioinformatics analysis to identify aberrant signaling pathways. We further performed molecular inhibition and silencing of the upstream regulatory kinase in gastric cancer cell lines and validated its effect on cellular phenotype. Through an ex vivo technology utilizing patient tumor and blood sample, we sought to understand the therapeutic potential of the kinase by recreating the tumor microenvironment. RESULTS: Using mass spectrometry-based high-throughput analysis, we identified 1,344 phosphosites and 848 phosphoproteins, including differential phosphorylation of 177 proteins (fold change cut-off ≥ 1.5). Our data showed that a subset of differentially phosphorylated proteins belonged to splicing machinery. Pathway analysis highlighted Cdc2-like kinase (CLK1) as upstream kinase. Inhibition of CLK1 using TG003 and CLK1 siRNA resulted in a decreased cell viability, proliferation, invasion and migration as well as modulation in the phosphorylation of SRSF2. Ex vivo experiments which utilizes patient's own tumor and blood to recreate the tumor microenvironment validated the use of CLK1 as a potential target for gastric cancer treatment. CONCLUSIONS: Our data indicates that CLK1 plays a crucial role in the regulation of splicing process in gastric cancer and that CLK1 can act as a novel therapeutic target in gastric cancer.

A draft map of the human proteome.

The availability of human genome sequence has transformed biomedical research over the past decade. However, an equivalent map for the human proteome with direct measurements of proteins and peptides does not exist yet. Here we present a draft map of the human proteome using high-resolution Fourier-transform mass spectrometry. In-depth proteomic profiling of 30 histologically normal human samples, including 17 adult tissues, 7 fetal tissues and 6 purified primary haematopoietic cells, resulted in identification of proteins encoded by 17,294 genes accounting for approximately 84% of the total annotated protein-coding genes in humans. A unique and comprehensive strategy for proteogenomic analysis enabled us to discover a number of novel protein-coding regions, which includes translated pseudogenes, non-coding RNAs and upstream open reading frames. This large human proteome catalogue (available as an interactive web-based resource at http://www.humanproteomemap.org) will complement available human genome and transcriptome data to accelerate biomedical research in health and disease.

Multi-Omics Driven Assembly and Annotation of the Sandalwood (Santalum album) Genome.

Indian sandalwood (Santalum album) is an important tropical evergreen tree known for its fragrant heartwood-derived essential oil and its valuable carving wood. Here, we applied an integrated genomic, transcriptomic, and proteomic approach to assemble and annotate the Indian sandalwood genome. Our genome sequencing resulted in the establishment of a draft map of the smallest genome for any woody tree species to date (221 Mb). The genome annotation predicted 38,119 protein-coding genes and 27.42% repetitive DNA elements. In-depth proteome analysis revealed the identities of 72,325 unique peptides, which confirmed 10,076 of the predicted genes. The addition of transcriptomic and proteogenomic approaches resulted in the identification of 53 novel proteins and 34 gene-correction events that were missed by genomic approaches. Proteogenomic analysis also helped in reassigning 1,348 potential noncoding RNAs as bona fide protein-coding messenger RNAs. Gene expression patterns at the RNA and protein levels indicated that peptide sequencing was useful in capturing proteins encoded by nuclear and organellar genomes alike. Mass spectrometry-based proteomic evidence provided an unbiased approach toward the identification of proteins encoded by organellar genomes. Such proteins are often missed in transcriptome data sets due to the enrichment of only messenger RNAs that contain poly(A) tails. Overall, the use of integrated omic approaches enhanced the quality of the assembly and annotation of this nonmodel plant genome. The availability of genomic, transcriptomic, and proteomic data will enhance genomics-assisted breeding, germplasm characterization, and conservation of sandalwood trees.

Multiomic analysis of oral keratinocytes chronically exposed to shisha.

BACKGROUND: Tobacco is smoked in different form including cigarettes and water pipes. One popular form of water pipe smoking especially in Middle Eastern countries is shisha smoking. Shisha has been associated with various diseases including oral cancer. However, genomic alterations and gene expression changes associated with chronic shisha exposure have not been previously investigated. OBJECTIVES: Whole-exome sequencing and gene expression profiling of immortalized human oral keratinocytes (OKF6/TERT1) cells chronically treated with 0.5% shisha extract for a period of 8 months was undertaken to characterize molecular alterations associated with shisha exposure. METHODS: Genomic DNA and RNA were extracted and preprocessed as per manufacturer's instruction and subjected to whole-exome and transcriptome sequencing using Illumina HiSeq2500 platform. Exome was analyzed using GATK pipeline whereas RNA-Seq data was analyzed using HiSat2 and HTSeq along with DESeq to elucidate differentially expressed genes. RESULTS: Whole-exome sequence analysis led to identification of 521 somatic missense variants corresponding to 389 genes RNA-Seq data revealed 247 differentially expressed genes (≥2-fold, P-value<0.01) in shisha treated cells compared to parental cells. Pathway analysis of differentially expressed genes revealed that interferon-signaling pathway was significantly affected. We predict activation of MAPK1 pathway which is known to play a key role in oral cancer. We also observed allele specific expression of mutant LIMA1 based on RNA-Seq dataset. CONCLUSION: Our findings provide insights into genomic alterations and gene expression pattern associated with oral keratinocytes chronically exposed to shisha.

SILAC-based quantitative proteomic analysis reveals widespread molecular alterations in human skin keratinocytes upon chronic arsenic exposure.

Chronic exposure to arsenic is associated with dermatological and nondermatological disorders. Consumption of arsenic-contaminated drinking water results in accumulation of arsenic in liver, spleen, kidneys, lungs, and gastrointestinal tract. Although arsenic is cleared from these sites, a substantial amount of residual arsenic is left in keratin-rich tissues including skin. Epidemiological studies suggest the association of skin cancer upon arsenic exposure, however, the mechanism of arsenic-induced carcinogenesis is not completely understood. We developed a cell line based model to understand the molecular mechanisms involved in arsenic-mediated toxicity and carcinogenicity. Human skin keratinocyte cell line, HaCaT, was chronically exposed to 100 nM sodium arsenite over a period of 6 months. We observed an increase in basal ROS levels in arsenic-exposed cells. SILAC-based quantitative proteomics approach resulted in identification of 2111 proteins of which 42 proteins were found to be overexpressed and 54 downregulated (twofold) upon chronic arsenic exposure. Our analysis revealed arsenic-induced overexpression of aldo-keto reductase family 1 member C2 (AKR1C2), aldo-keto reductase family 1 member C3 (AKR1C3), glutamate-cysteine ligase catalytic subunit (GCLC), and NAD(P)H dehydrogenase [quinone] 1 (NQO1) among others. We observed downregulation of several members of the plakin family including periplakin (PPL), envoplakin (EVPL), and involucrin (IVL) that are essential for terminal differentiation of keratinocytes. MRM and Western blot analysis confirmed differential expression of several candidate proteins. Our study provides insights into molecular alterations upon chronic arsenic exposure on skin.

Quantitative Proteomic and Phosphoproteomic Analysis of H37Ra and H37Rv Strains of Mycobacterium tuberculosis.

Mycobacterium tuberculosis, the causative agent of tuberculosis, accounts for 1.5 million human deaths annually worldwide. Despite efforts to eradicate tuberculosis, it still remains a deadly disease. The two best characterized strains of M. tuberculosis, virulent H37Rv and avirulent H37Ra, provide a unique platform to investigate biochemical and signaling pathways associated with pathogenicity. To delineate the biomolecular dynamics that may account for pathogenicity and attenuation of virulence in M. tuberculosis, we compared the proteome and phosphoproteome profiles of H37Rv and H37Ra strains. Quantitative phosphoproteomic analysis was performed using high-resolution Fourier transform mass spectrometry. Analysis of exponential and stationary phases of these strains resulted in identification and quantitation of 2709 proteins along with 512 phosphorylation sites derived from 257 proteins. In addition to confirming the presence of previously described M. tuberculosis phosphorylated proteins, we identified 265 novel phosphorylation sites. Quantitative proteomic analysis revealed more than five-fold upregulation of proteins belonging to virulence associated type VII bacterial secretion system in H37Rv when compared to those in H37Ra. We also identified 84 proteins, which exhibited changes in phosphorylation levels between the virulent and avirulent strains. Bioinformatics analysis of the proteins altered in their level of expression or phosphorylation revealed enrichment of pathways involved in fatty acid biosynthesis and two-component regulatory system. Our data provides a resource for further exploration of functional differences at molecular level between H37Rv and H37Ra, which will ultimately explain the molecular underpinnings that determine virulence in tuberculosis.

Synovial fluid proteome in rheumatoid arthritis.

BACKGROUND: Rheumatoid arthritis (RA) is a chronic autoinflammatory disorder that affects small joints. Despite intense efforts, there are currently no definitive markers for early diagnosis of RA and for monitoring the progression of this disease, though some of the markers like anti CCP antibodies and anti vimentin antibodies are promising. We sought to catalogue the proteins present in the synovial fluid of patients with RA. It was done with the aim of identifying newer biomarkers, if any, that might prove promising in future. METHODS: To enrich the low abundance proteins, we undertook two approaches-multiple affinity removal system (MARS14) to deplete some of the most abundant proteins and lectin affinity chromatography for enrichment of glycoproteins. The peptides were analyzed by LC-MS/MS on a high resolution Fourier transform mass spectrometer. RESULTS: This effort was the first total profiling of the synovial fluid proteome in RA that led to identification of 956 proteins. From the list, we identified a number of functionally significant proteins including vascular cell adhesion molecule-1, S100 proteins, AXL receptor protein tyrosine kinase, macrophage colony stimulating factor (M-CSF), programmed cell death ligand 2 (PDCD1LG2), TNF receptor 2, (TNFRSF1B) and many novel proteins including hyaluronan-binding protein 2, semaphorin 4A (SEMA4D) and osteoclast stimulating factor 1. Overall, our findings illustrate the complex and dynamic nature of RA in which multiple pathways seems to be participating actively. CONCLUSIONS: The use of high resolution mass spectrometry thus, enabled identification of proteins which might be critical to the progression of RA.

Annotation of the zebrafish genome through an integrated transcriptomic and proteomic analysis.

Accurate annotation of protein-coding genes is one of the primary tasks upon the completion of whole genome sequencing of any organism. In this study, we used an integrated transcriptomic and proteomic strategy to validate and improve the existing zebrafish genome annotation. We undertook high-resolution mass-spectrometry-based proteomic profiling of 10 adult organs, whole adult fish body, and two developmental stages of zebrafish (SAT line), in addition to transcriptomic profiling of six organs. More than 7,000 proteins were identified from proteomic analyses, and ∼ 69,000 high-confidence transcripts were assembled from the RNA sequencing data. Approximately 15% of the transcripts mapped to intergenic regions, the majority of which are likely long non-coding RNAs. These high-quality transcriptomic and proteomic data were used to manually reannotate the zebrafish genome. We report the identification of 157 novel protein-coding genes. In addition, our data led to modification of existing gene structures including novel exons, changes in exon coordinates, changes in frame of translation, translation in annotated UTRs, and joining of genes. Finally, we discovered four instances of genome assembly errors that were supported by both proteomic and transcriptomic data. Our study shows how an integrative analysis of the transcriptome and the proteome can extend our understanding of even well-annotated genomes.

Proteomic Signature of Endothelial Dysfunction Identified in the Serum of Acute Ischemic Stroke Patients by the iTRAQ-Based LC-MS Approach.

Acute ischemic stroke (AIS) is a devastating cerebrovascular disorder that leads to permanent physical and neurological disabilities in adults worldwide. Proteins associated with stroke pathogenesis may appear in the serum of AIS patients due to blood-brain barrier dysfunction, thus permitting the development of blood-based biomarkers for early diagnosis of stroke. These biomarkers could perhaps be an adjunct to the existing imaging modalities and aid in better management and therapeutic intervention during the course of the disease. For this exploratory study, a combination of multiplexed isobaric tagging using iTRAQ reagents and high resolution tandem mass spectrometry was used to identify differentially expressed proteins in serum samples from AIS patients. The quantitative proteomic analysis of serum from both AIS and control subjects revealed 389 high confidence protein identifications and their relative levels. Among them, 60 proteins showed a ≥1.5-fold change in the AIS subjects. We verified the altered serum levels of candidate proteins such as vWF, ADAMTS13, S100A7, and DLG4 through ELISA, and the results also corroborate with the experimental findings. vWF and ADAMTS13 are key players that regulate blood hemostasis, and their altered concentration may contribute to endothelial dysfunction. S100A7 is a novel candidate protein identified in this study that is also known to mediate inflammation, endothelial proliferation, and angiogenesis. The current study provided a potential and novel biomarker panel that may in turn provide diagnostic aid to the existing imaging modalities for the rapid diagnosis of ischemic stroke.

Chromosome-centric human proteome project: deciphering proteins associated with glioma and neurodegenerative disorders on chromosome 12.

In line with the aims of the Chromosome-centric Human Proteome Project (C-HPP) to completely annotate proteins of each chromosome and biology/disease driven HPP (B/D-HPP) to decipher their relation to diseases, we have generated a nonredundant catalogue of protein-coding genes for Chromosome 12 (Chr. 12) and further annotated proteins associated with major neurological disorders. Integrating high level proteomic evidence from four major databases (neXtProt, Global Proteome Machine (GPMdb), PeptideAtlas, and Human Protein Atlas (HPA)) along with Ensembl data resource resulted in the identification of 1066 protein coding genes, of which 171 were defined as "missing proteins" based on the weak or complete absence of experimental evidence. With functional annotations using DAVID and GAD, about 40% of the proteins could be grouped as brain related with implications in cancer or neurological disorders. We used published and unpublished high confidence mass spectrometry data from our group and other literature consisting of more than 5000 proteins derived from clinical specimens from patients with human gliomas, Alzheimer's disease, and Parkinson's disease and mapped it onto Chr. 12. We observed a total of 202 proteins mapping to human Chr. 12, 136 of which were differentially expressed in these disease conditions as compared to the normal. Functional grouping indicated their association with cell cycle, cell-to-cell signaling, and other important processes and networks, whereas their disease association analysis confirmed neurological diseases and cancer as the major group along with psycological disorders, with several overexpressed genes/proteins mapping to 12q13-15 amplicon region. Using multiple strategies and bioinformatics tools, we identified 103 differentially expressed proteins to have secretory potential, 17 of which have already been reported in direct analysis of the plasma or cerebrospinal fluid (CSF) from the patients and 21 of them mapped to cancer associated protein (CAPs) database that are amenable to selective reaction monitoring (SRM) assays for targeted proteomic analysis. Our analysis also reveals, for the first time, mass spectrometric evidence for two "missing proteins" from Chr. 12, namely, synaptic vesicle 2-related protein (SVOP) and IQ motif containing D (IQCD). The analysis provides a snapshot of Chr. 12 encoded proteins associated with gliomas and major neurological conditions and their secretability which can be used to drive efforts for clinical applications.

Identification of prosaposin and transgelin as potential biomarkers for gallbladder cancer using quantitative proteomics.

Gallbladder cancer is an uncommon but lethal malignancy with particularly high incidence in Chile, India, Japan and China. There is a paucity of unbiased large-scale studies investigating molecular basis of gallbladder cancer. To systematically identify differentially regulated proteins in gallbladder cancer, iTRAQ-based quantitative proteomics of gallbladder cancer was carried out using Fourier transform high resolution mass spectrometry. Of the 2575 proteins identified, proteins upregulated in gallbladder cancer included several lysosomal proteins such as prosaposin, cathepsin Z and cathepsin H. Downregulated proteins included serine protease HTRA1 and transgelin, which have been reported to be downregulated in several other cancers. Novel biomarker candidates including prosaposin and transgelin were validated to be upregulated and downregulated, respectively, in gallbladder cancer using tissue microarrays. Our study provides the first large scale proteomic characterization of gallbladder cancer which will serve as a resource for future discovery of biomarkers for gallbladder cancer.

QTL mapping of human retina DNA methylation identifies 87 gene-epigenome interactions in age-related macular degeneration.

DNA methylation provides a crucial epigenetic mark linking genetic variations to environmental influence. We have analyzed array-based DNA methylation profiles of 160 human retinas with co-measured RNA-seq and >8 million genetic variants, uncovering sites of genetic regulation in cis (37,453 methylation quantitative trait loci and 12,505 expression quantitative trait loci) and 13,747 DNA methylation loci affecting gene expression, with over one-third specific to the retina. Methylation and expression quantitative trait loci show non-random distribution and enrichment of biological processes related to synapse, mitochondria, and catabolism. Summary data-based Mendelian randomization and colocalization analyses identify 87 target genes where methylation and gene-expression changes likely mediate the genotype effect on age-related macular degeneration. Integrated pathway analysis reveals epigenetic regulation of immune response and metabolism including the glutathione pathway and glycolysis. Our study thus defines key roles of genetic variations driving methylation changes, prioritizes epigenetic control of gene expression, and suggests frameworks for regulation of macular degeneration pathology by genotype-environment interaction in retina.

Integrating genetic regulation and single-cell expression with GWAS prioritizes causal genes and cell types for glaucoma.

Primary open-angle glaucoma (POAG), characterized by retinal ganglion cell death, is a leading cause of irreversible blindness worldwide. However, its molecular and cellular causes are not well understood. Elevated intraocular pressure (IOP) is a major risk factor, but many patients have normal IOP. Colocalization and Mendelian randomization analysis of >240 POAG and IOP genome-wide association study (GWAS) loci and overlapping expression and splicing quantitative trait loci (e/sQTLs) in 49 GTEx tissues and retina prioritizes causal genes for 60% of loci. These genes are enriched in pathways implicated in extracellular matrix organization, cell adhesion, and vascular development. Analysis of single-nucleus RNA-seq of glaucoma-relevant eye tissues reveals that the POAG and IOP colocalizing genes and genome-wide associations are enriched in specific cell types in the aqueous outflow pathways, retina, optic nerve head, peripapillary sclera, and choroid. This study nominates IOP-dependent and independent regulatory mechanisms, genes, and cell types that may contribute to POAG pathogenesis.

A multilectin affinity approach for comparative glycoprotein profiling of rheumatoid arthritis and spondyloarthropathy.

BACKGROUND: Arthritis refers to inflammation of joints and includes common disorders such as rheumatoid arthritis (RA) and spondyloarthropathies (SpAs). These diseases differ mainly in terms of their clinical manifestations and the underlying pathogenesis. Glycoproteins in synovial fluid might reflect the disease activity status in the joints affected by arthritis; yet they have not been systematically studied previously. Although markers have been described for assisting in the diagnosis of RA, there are currently no known biomarkers for SpA. MATERIALS AND METHODS: We sought to determine the relative abundance of glycoproteins in RA and SpA by lectin affinity chromatography coupled to iTRAQ labeling and LC-MS/MS analysis. We also used ELISA to validate the overexpression of VCAM-1, one of the candidate proteins identified in this study, in synovial fluid from RA patients. RESULTS AND DISCUSSION: We identified proteins that were previously reported to be overexpressed in RA including metalloproteinase inhibitor 1 (TIMP1), myeloperoxidase (MPO) and several S100 proteins. In addition, we discovered several novel candidates that were overexpressed in SpA including Apolipoproteins C-II and C-III and the SUN domain-containing protein 3 (SUN3). Novel molecules found overexpressed in RA included extracellular matrix protein 1 (ECM1) and lumican (LUM). We validated one of the candidate biomarkers, vascular cell adhesion molecule 1 (VCAM1), in 20 RA and SpA samples using ELISA and confirmed its overexpression in RA (p-value <0.01). Our quantitative glycoproteomic approach to study arthritic disorders should open up new avenues for additional proteomics-based discovery studies in rheumatological disorders.

QTL mapping of human retina DNA methylation identifies 87 gene-epigenome interactions in age-related macular degeneration.

DNA methylation (DNAm) provides a crucial epigenetic mark linking genetic variations to environmental influence. We analyzed array-based DNAm profiles of 160 human retinas with co-measured RNA-seq and > 8 million genetic variants, uncovering sites of genetic regulation in cis (37,453 mQTLs and 12,505 eQTLs) and 13,747 eQTMs (DNAm loci affecting gene expression), with over one-third specific to the retina. mQTLs and eQTMs show non-random distribution and enrichment of biological processes related to synapse, mitochondria, and catabolism. Summary data-based Mendelian randomization and colocalization analyses identify 87 target genes where methylation and gene-expression changes likely mediate the genotype effect on age-related macular degeneration (AMD). Integrated pathway analysis reveals epigenetic regulation of immune response and metabolism including the glutathione pathway and glycolysis. Our study thus defines key roles of genetic variations driving methylation changes, prioritizes epigenetic control of gene expression, and suggests frameworks for regulation of AMD pathology by genotype-environment interaction in retina.