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Glycans are an essential structural component of Immunoglobulin G (IgG) that modulate its structure and function. However, regulatory mechanisms behind this complex posttranslational modification are not well known. Previous genome-wide association studies (GWAS) identified 29 genomic regions involved in regulation of IgG glycosylation, but only a few were functionally validated. One of the key functional features of IgG glycosylation is the addition of galactose (galactosylation). We performed GWAS of IgG galactosylation (N=13,705) and identified 16 significantly associated loci, indicating that IgG galactosylation is regulated by a complex network of genes that extends beyond the galactosyltransferase enzyme that adds galactose to IgG glycans. Gene prioritization identified 37 candidate genes. Using a recently developed CRISPR/dCas9 system we manipulated gene expression of candidate genes in thein vitroIgG expression system. Up- and downregulation of three genes,EEF1A1, MANBAandTNFRSF13B, changed the IgG glycome composition, which confirmed that these three genes are involved in IgG galactosylation in thisin vitroexpression system.

Supplementary Table 1. Directly measured glycans and derived glycan traits; Supplementary Table 2. IgG glycome composition in CRC patients and controls; Supplementary Table 3. Pleiotropic effects of top glycan SNPs relevant for fucosylation on CRC.

Purpose: Alternative glycosylation has significant structural and functional consequences on IgG and consequently also on cancer immunosurveillance. Because of technological limitations, the effects of highly heritable individual variations and the differences in the dynamics of changes in IgG glycosylation on colorectal cancer were never investigated before.Experimental Design: Using recently developed high-throughput UPLC technology for IgG glycosylation analysis, we analyzed IgG glycome composition in 760 patients with colorectal cancer and 538 matching controls. Effects of surgery were evaluated in 28 patients sampled before and three times after surgery. A predictive model was built using regularized logistic regression and evaluated using a 10-cross validation procedure. Furthermore, IgG glycome composition was analyzed in 39 plasma samples collected before initial diagnosis of colorectal cancer.Results: We have found that colorectal cancer associates with decrease in IgG galactosylation, IgG sialylation and increase in core-fucosylation of neutral glycans with concurrent decrease of core-fucosylation of sialylated glycans. Although a model based on age and sex did not show discriminative power (AUC = 0.499), the addition of glycan variables into the model considerably increased the discriminative power of the model (AUC = 0.755). However, none of these differences were significant in the small set of samples collected before the initial diagnosis.Conclusions: Considering the functional relevance of IgG glycosylation for both tumor immunosurveillance and clinical efficacy of therapy with mAbs, individual variation in IgG glycosylation may turn out to be important for prediction of disease course or the choice of therapy, thus warranting further, more detailed studies of IgG glycosylation in colorectal cancer. Clin Cancer Res; 22(12); 3078–86. ©2016 AACR.

Alternative glycosylation of immunoglobulin G (IgG) is functionally important in multiple human physiological and pathological states. Our understanding of molecular mechanisms that regulate IgG glycosylation is vague because of the complexity of this process, which involves hundreds of genes. Several genome-wide association (GWA) studies have revealed a network of genes associated with IgG glycosylation that are pleiotropic for a number of diseases. Here, we report a design of a versatile system for IgG production and gene manipulations that can be used for in vitro functional follow-up of GWA hits or any gene of interest. The system is based on CRISPR-dCas9, extended by a piggyBac integrase compatible vector, and drives IgG production in HEK-293F cells. We validated our systems that stably express VPR-dCas9 and KRAB-dCas9 by manipulation of four glyco-genes with a known role in IgG glycosylation, and then functionally validated three GWAS hits for IgG glycosylation with an as-yet-unknown role in this process.

Many recent epigenetic studies utilize the advantages of CRISPR/dCas9 based tools in linking certain epigenetic modification with gene expression regulation. Various multifactorial diseases often contain changed epigenetic signatures at many loci, so tools for simultaneously targeting different loci would significantly facilitate the understanding of disease pathogenesis. We tested different dCas9 orthologs (dCjCas9, dNmCas9, dSt1Cas9, dFnCas9, dSaCas9 and dSpCas9) in C-terminal fusion with DNMT3A effector domain to find candidates that potentiate effector domain to perform its function at the target site. We demonstrated that nuclear localization signals (NLS) at both termini of fusion constructs is crucial for both proper nuclear import of such large constructs as well as for maximization of targeted DNA methylation activity. We identified SpCas9, SaCas9 and CjCas9 as potential candidates for the fusion constructs. With further optimization of the SaCas9 ortholog, due to less complex PAM requirements in contrast to CjCas9, we showed that N-terminal fusion with DNMT3A (dSaCas9- DNMT3A) is optimal to exert targeted DNA methylation activity comparable to the dSpCas9- DNMT3A construct. N-terminal fusions showed better results for both Cas9 orthologs, SaCas9 and SpCas9, so it can be used as universal approach for linking different effector domains in order to obtain highly active fusions.

Glycosylation of immunoglobulin G (IgG) largely defines function of this antibody and regulates inflammation on multiple levels but the mechanisms and the genes involved in the process of IgG glycosylation are still vaguely understood. Genome wide association studies (GWAS) enabled identification of many genes potentially involved in IgG glycosylation but in the most cases functional validation is missing. Composition of the IgG glycome changes significantly with various diseases and ageing. In women, the most prominent change occurs during the perimenopausal period and a recent intervention study revealed that estrogen regulates IgG glycosylation. The most affected IgG glycome features are galactosylation, sialylation and structures with bisecting GlcNac. Analysis of the Signaling Pathway Projects (SPP) web knowledgebase revealed that E2 affects expression of four genes with yet unknown role in IgG glycosylation with three of them associated with galactosylation (RUNX1, RUNX3 and SPINK4) and one associated with sialylation (ELL2) through previous GWA studies. To map downstream signaling mechanisms responsible for the effect of estrogen on IgG glycome composition we utilized our recently developed in vitro IgG expression system HEK293-FS for targeted CRISPRa/CRISPRi manipulation of candidate loci. This system exploits stably integrated dCas9-VPR or dCas9-KRAB expression cassettes for targeted upregulation or downregulation of genes via transient transfection of FreeStyleTM 293-F cells with specific gRNAs. We successfully upregulated or downregulated RUNX1, RUNX3, SPINK4 and ELL2 but only upregulation of RUNX3 (Runt-related factor 3) and SPINK4 (Serine Peptidase Inhibitor Kazal Type 4) resulted with concomitant change in IgG glycome composition. Upregulation of RUNX3 resulted with significant decrease in galactosylated glycans accompanied with increase in agalactosylated glycans. Upregulation of SPINK4 was also accompanied with decrease in galactosylated glycans but the ratio of agalactosylated glycans remained stable. To further explore the role of SPINK4 and RUNX3 in IgG galactosylation we measured B4GALT1 (beta-1, 4- galactosyltransferase 1) expression after upregulation and observed no significant change in B4GALT1 transcript levels. This suggests that RUNX3 and SPINK4 do not suppress B4GALT1 on transcription level, but through some indirect pathway that should be further investigated. Taken together, the results of this study suggest the molecular mechanism through which E2 could regulate IgG glycosylation, specifically galactosylation.

Pro- or anti-inflammatory activity of immunoglobulin G (IgG) depends largely on differential N- glycosylation of its Fc region. Alternative glycosylation affects binding efficiency of this antibody to antigens, ligands, or different complexes of the immune system, which modulates immune response. While previous genome-wide association studies (GWAS) succeeded to associate functional network of gene loci with IgG glycosylation pleiotropic with inflammatory diseases, it is still unclear whether these genes have a functional importance and if so, how they are involved in this process. For this purpose, we applied already established system based on FreeStyle™ 293-F cells, with stably integrated CRISPR/dCas9 fusions (dCas9-VPR or dCas9-KRAB) for direct regulation of the candidate genes using appropriate sgRNAs. The cell system is designed to secrete IgG molecules, so that glycans can be analyzed as the final phenotype following gene manipulations. The GWA hits were grouped with respect to different glycosylation traits such as galactosylation (KIF3C, NFKB1, MANBA, SLC38A10, TNFRSF13B, EEF1A1, HIVEP2), fucosylation (MYCBP, ACVR1C, KIF11, TBX21, TBKBP1, NFATC2), sialylation (IL6ST, GGA2, SLC17A9, SPPL3) and bisecting GlcNAC (TCF3, SMARCB1, DERL2, RRBP1, KDELR2). Out of seven genes associated with galactysylation only MANBA, NFKB1, TNFRSF13B and EEF1A1 showed change of agalactosylated structures when upregulated using dCas9-VPR. Out of six hits associated with fucosylation, only upregulation of TBX21 and TBKBP1 showed significant changes in galactosylation, but we did not detect any changes in fucosylation. This can suggest that a different cell model might be preferable to validate if these GWA hits are responsible for fucosylation, since glycans on IgG produced from FreeStyle™ 293-F cells possess only core fucose. Out of five loci associated with bisecting GlcNac, only the upregulation of KDELR2 produced an increase in biantennary glycan structures with bisecting GlcNac. The upregulation of DERL2 and RRBP1 resulted in an increase of digalactosylated biantennary glycans. Out of four GWA hits for sialylation, only downregulation of SPPL3 led to hyperglycoslation with the corresponding increase in sialylated and galactosylated structures and decrease in agalactosylated glycans, even though all the genes were successfully up - or downregulated. In conclusion, in our cell model, which produces human plasma-like IgG, we have proven the functional role of several GWA hits which are not glycosyltransferases but are associated with the IgG glycosylation pathway. Ongoing research will unravel the exact role of these genes in an integrated picture of molecular mechanism regulating IgG glycosylation.

Due to the limited therapeutic options after ischemic stroke, gene therapy has emerged as a promising choice, especially with recent advances in viral vector delivery systems. Therefore, we aimed to provide the current state of the art of lentivirus (LV) and adeno-associated virus (AAV) mediated gene interventions in preclinical ischemic stroke models. A systematic analysis including qualitative and quantitative syntheses of studies published until December 2020 was performed. Most of the 87 selected publications used adult male rodents and the preferred stroke model was transient middle cerebral artery occlusion. LV and AAV vectors were equally used for transgene delivery, however loads of AAVs were higher than LVs. Serotypes having broad cell tropism, the use of constitutive promoters, and virus delivery before the stroke induction via stereotaxic injection in the cortex and striatum were preferred in the analyzed studies. The meta-analysis based on infarct volume as the primary outcome confirmed the efficacy of the preclinical interventions. The quality assessment exposed publication bias and setbacks in regard to risks of bias and study relevance. The translational potential could increase by using specific cell targeting, post-stroke interventions, non-invasive systematic delivery, and use of large animals.

Alternative glycosylation of immunoglobulin G (IgG) is functionally important in multiple human physiological and pathological states. One of the best examples is a simple fucosylation that can switch IgG effector function between pro- and anti- inflammatory, which is critical in production of therapeutic antibodies. Several genome-wide association studies revealed a network of genes associated with IgG glycosylation which are pleiotropic for several diseases. To understand the complex molecular mechanisms that regulate IgG glycosylation, we designed a versatile system for IgG production and gene manipulation that can be used for in vitro functional follow-up of GWA hits or any gene of interest. The system is based on CRISPR/dCas9, extended by a piggyBac integrase compatible vector, and drives IgG production in HEK-293F cells, essentially creating derived cell lines with integrated machinery for programmed transcriptional control. Transient transfection of those cells with the second component of the system, a plasmid for monocistronic expression of both IgG chains and co-expression of gRNA molecules, enables the readout of IgG glycan profiles following precisely targeted up- or downregulation of genes of interest. Though the development of the system was complex, its utilization by the end user is simple and straightforward. Transfection of the large dCas9 fusion constructs is quite inefficient and represents a bottleneck in effective experimental design with high throughput. The bipartite nature of the system ensures that only the cells expressing gRNA (with concomitant transcriptional alteration) produce IgG that is analyzed for glycosylation profile. We validated our derived cell lines that stably express VPR-dCas9 and KRAB-dCas9 by manipulation of four glycogenes with a known role in IgG glycosylation. Transcriptional up- and downregulation of B4GALT1, encoding the enzyme that adds galactose to the core glycan structure, had the predictable effect on the abundance of agalactosylated and galactosylated structures, changing the glycosylation profile depending on the transcript level. Similarly, downregulation of FUT8, encoding a fucosyltransferase, decreased the core-fucosylated structures, while upregulation of ST6GAL1 and MGAT3 increased sialylated structures and those with a bisecting GlcNAc, respectively, which was in line with the expected change in enzyme levels. Interestingly, the change in sialylated structures was accompanied by changes in galactosylation, again demonstrating the complexity of the IgG glycosylation network. Our newly developed system for transient expression of can be used for studying the role of genes associated with IgG glycosylation by GWA studies. It can also be easily repurposed to serve as a model for other proteins and their posttranslational modifications, with appropriate targeting via gRNA.

Establishing causal relationship between epigenetic marks and gene transcription requires molecular tools, which can precisely modify specific genomic regions. Here, we present a modular and extensible CRISPR/dCas9-based toolbox for epigenetic editing and direct gene regulation. It features a system for expression of orthogonal dCas9 proteins fused to various effector domains and includes a multi-gRNA system for simultaneous targeting dCas9 orthologs to up to six loci. The C- and N-terminal dCas9 fusions with DNMT3A and TET1 catalytic domains were thoroughly characterized. We demonstrated simultaneous use of the DNMT3A-dSpCas9 and TET1-dSaCas9 fusions within the same cells and showed that imposed cytosine hyper- and hypo-methylation altered level of gene transcription if targeted CpG sites were functionally relevant. Dual epigenetic manipulation of the HNF1A and MGAT3 genes, involved in protein N-glycosylation, resulted in change of the glycan phenotype in BG1 cells. Furthermore, simultaneous targeting of the TET1-dSaCas9 and VPR-dSpCas9 fusions to the HNF1A regulatory region revealed strong and persistent synergistic effect on gene transcription, up to 30 days following cell transfection, suggesting involvement of epigenetic mechanisms in maintenance of the reactivated state. Also, modulation of dCas9 expression effectively reduced off-target effects while maintaining the desired effects on target regions.

Molecular tools for gene regulation and epigenome editing consist of two main parts: the targeting moiety binding a specific genomic locus and the effector domain performing the editing or regulatory function. The advent of CRISPR-Cas9 technology enabled easy and flexible targeting of almost any locus by co-expression of a small sgRNA molecule, which is complementary to the target sequence and forms a complex with Cas9, directing it to that particular target. Here, we review strategies for recruitment of effector domains, used in gene regulation and epigenome editing, to the dCas9 DNA-targeting protein. To date, the most important CRISPR-Cas9 applications in gene regulation are CRISPR activation or interference, while epigenome editing focuses on targeted changes in DNA methylation and histone modifications. Several strategies for signal amplification by recruitment of multiple effector domains deserve special focus. While some approaches rely on altering the sgRNA molecule and extending it with aptamers for effector domain recruitment, others use modifications to the Cas9 protein by direct fusions with effector domains or by addition of an epitope tag, which also has the ability to bind multiple effector domains. A major barrier to the widespread use of CRISPR-Cas9 technology for therapeutic purposes is its off-target effect. We review efforts to enhance CRISPR-Cas9 specificity by selection of Cas9 orthologs from various bacterial species and their further refinement by introduction of beneficial mutations. The molecular tools available today enable a researcher to choose the best balance of targeting flexibility, activity amplification, delivery method and specificity.

Epigenetic studies relied so far on correlations be- tween epigenetic marks and gene expression pat- tern. Technologies developed for epigenome edit- ing now enable direct study of functional relevance of precise epigenetic modifications and gene reg- ulation. The reversible nature of epigenetic modi- fications, including DNA methylation, has been al- ready exploited in cancer therapy for remodeling the aberrant epigenetic landscape. However, this was achieved non-selectively using epigenetic inhibitors. Epigenetic editing at specific loci represents a novel approach that might selectively and heritably alter gene expression. Here, we developed a CRISPR- Cas9-based tool for specific DNA methylation con- sisting of deactivated Cas9 (dCas9) nuclease and cat- alytic domain of the DNA methyltransferase DNMT3A targeted by co–expression of a guide RNA to any 20 bp DNA sequence followed by the NGG trinucleotide. We demonstrated targeted CpG methylation in a ∼35 bp wide region by the fusion protein. We also showed that multiple guide RNAs could target the dCas9- DNMT3A construct to multiple adjacent sites, which enabled methylation of a larger part of the promoter. DNA methylation activity was specific for the tar- geted region and heritable across mitotic divisions. Finally, we demonstrated that directed DNA methy- lation of a wider promoter region of the target loci IL6ST and BACH2 decreased their expression.

Background: Many genome- and epigenome-wide association studies (GWAS and EWAS) and studies of promoter methylation of candidate genes for inflammatory bowel disease (IBD) have demonstrated significant associations between genetic and epigenetic changes and IBD. Independent GWA studies have identified genetic variants in the BACH2, IL6ST, LAMB1, IKZF1, and MGAT3 loci to be associated with both IBD and immunoglobulin G (IgG) glycosylation. Methods: Using bisulfite pyrosequencing, we analyzed CpG methylation in promoter regions of these five genes from peripheral blood of several hundred IBD patients and healthy controls (HCs) from two independent cohorts, respectively. Results: We found significant differences in the methylation levels in the MGAT3 and BACH2 genes between both Crohn's disease and ulcerative colitis when compared to HC. The same pattern of methylation changes was identified for both genes in CD19 + B cells isolated from the whole blood of a subset of the IBD patients. A correlation analysis was performed between the MGAT3 and BACH2 promoter methylation and individual IgG glycans, measured in the same individuals of the two large cohorts. MGAT3 promoter methylation correlated significantly with galactosylation, sialylation, and bisecting GlcNAc on IgG of the same patients, suggesting that activity of the GnT-III enzyme, encoded by this gene, might be altered in IBD. The correlations between the BACH2 promoter methylation and IgG glycans were less obvious, since BACH2 is not a glycosyltransferase and therefore may affect IgG glycosylation only indirectly. Conclusions: Our results suggest that epigenetic deregulation of key glycosylation genes might lead to an increase in pro-inflammatory properties of IgG in IBD through a decrease in galactosylation and sialylation and an increase of bisecting GlcNAc on digalactosylated glycan structures. Finally, we showed that CpG methylation in the promoter of the MGAT3 gene is altered in CD3 + T cells isolated from inflamed mucosa of patients with ulcerative colitis from a third smaller cohort, for which biopsies were available, suggesting a functional role of this glyco-gene in IBD pathogenesis. The authors would like to thank Stephanie Scott for her organizational and administrational contribution. The study has been funded by the EU FP7 grant European Commission IBD-BIOM (contract # 305479), EU FP7 Regional Potential Grant INTEGRA-Life (contract # 315997), European Structural and Investment Funds grant for the Croatian National Centre of Research Excellence in Personalized Healthcare (contract # KK.01.1.1.01.0010), and Croatian Science Foundation grant EpiGlycoIgG (contract # 3361). Financial support from Portugal (PI: SSP): FEDER—Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020—Operacional Programme for Competitiveness and Internationalisation (POCI), Portugal 2020, and by Portuguese funds through FCT—Fundação para a Ciência e a Tecnologia/ Ministério da Ciência, Tecnologia e Inovação in the framework of the project (POCI-01/0145-FEDER-016601; PTDC/DTP-PIC/0560/2014) was received. SSP also acknowledges the European Crohn’s and Colitis Organization (ECCO) and the “Broad Medical Research program at Crohn’s and Colitis Foundation of America-CCFA” for funding. SSP acknowledges the Portuguese Group of Study on IBD (GEDII) for funding. A.M.D. [PD/BD/105982/2014] also acknowledges FCT for funding. IBD-BIOM consortium: Daniel Kolarich (Department of Biomolecular Systems, Max Planck Institute of Colloids and Interfaces, Potsdam, Germany), Manfred Wuhrer (Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, The Netherlands; Division of BioAnalytical Chemistry, VU University Amsterdam, Amsterdam, the Netherlands), Dermot P. B. McGovern (F. Widjaja Family Foundation Inflammatory Bowel and Immunobiology Research Institute, Cedars-Sinai Medical Center, Los Angeles), Iain K. Pemberton (IP Research Consulting SAS, Paris, France), Daniel IR Spencer (Ludger Ltd., Culham Science Centre, Oxford, UK, Daryl L. Fernandes (Ludger Ltd., Culham Science Centre, Oxford, UK), Rahul Kalla, Kate O’Leary, Alex T Adams, Hazel Drummond, Elaine Nimmo, Ray Boyapati, David C Wilson (Centre for Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK), Ray Doran (Ludger Ltd., Culham Science Centre, Oxford, UK), Igor Rudan (all, Centre for Population Health Sciences, University of Edinburgh, Edinburgh, UK), Paolo Lionetti (Paediatric Gastroenterology Unit, AOU Meyer, Viale Pieraccini, Florence, Italy), Natalia Manetti (Department of Medical and Surgical Sciences, Division of Gastroenterology, University Hospital Careggi, Florence, Italy), Fabrizio Bossa (Department of Medical Sciences, Division of Gastroenterology, IRCCS-CSS Hospital, Viale Cappuccini, Rotondo, Italy), Paola Cantoro, Anna Kohn (Division of Gastroenterology, S. Camillo Hospital, Rome, Italy), Giancarlo Sturniolo (Gastrointestinal Unit, University of Padua, Padua, Italy), Silvio Danese (IBD Unit, Humanitas Research Institute, Rozzano, Milan, Italy), Mariek Pierik (Maastricht University Medical Centre (MUMC), Maastricht, the Netherlands), and David C. Wilson (Centre for Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK). This independent research was generously supported by the following grants: EU FP7 research grant IBD-BIOM (contract # 305479) to JS, VA, GL, and VZ; EU FP7 Regional Potential Grant INTEGRA-Life (contract # 315997) to GL and VZ; European Structural and Investment Funds grant for the Croatian National Centre of Research Excellence in Personalized Healthcare (contract # KK.01.1.1.01.0010) to GL and VZ; Croatian Science Foundation grant EpiGlycoIgG (contract # 3361) to VZ; FEDER COMPETE 2020 POCI, Portugal 2020, and Portuguese funds through FCT (contracts # POCI-01/0145-FEDER-016601 and PTDC/DTP-PIC/0560/2014) to SP; and FTC (contract # PD/BD/105982/2014) to AMD.

The incidence of perinatal mortality of Arabian horses in Croatia is in accordance with the high frequency of lethal and sublethal mutations in Arabian horses in other countries. The objective of this study was to investigate the frequency of recessive genes causing lavender foal syndrome (LFS) in three Croatian breeds of Arabian horse and to compare these findings with the literature reports. Pedigree and molecular analyses indicated the existence of three distinct breeds of Arabian horses (Traditional Arabian, Shagya Arab, and Pure Arabian horse) in Croatia. Molecular analyses were performed on 100 blood samples (n = 100) of Arabian horses by analyzing isolated and quality-tested genomic DNA. Polymerase chain reaction-restriction fragment length polymorphism analysis of the MYO5A gene was performed for LFS determination. The proportion of heterozygous carriers and the frequency of the LFS allele were lower in Arabian horse breeds in Croatia than in other reports. The LFS allele was found exclusively in the Pure Arabian breed, supporting the findings of other studies on Egyptian Arabian horses. The results are consistent with the interbreeding of Pure Arabian horses in Croatia with Egyptian Arabian horses. The Traditional Arabian and Shagya Arab breeds showed a lower frequency of the LFS allele. Based on these results, screening of the entire population of Arabian horses in Croatia for mutations responsible for multiple inherited genetic disorders should be included in breeding programs.

Background The RAS association domain family protein 1a (RASSF1A) is a prominent tumor suppressor gene showing altered promoter methylation in testicular germ cell tumors (TGCT). RASSF1A promoter hypermethylation might represent an early event in TGCT tumorigenesis. We investigated whether the RASSF1A promoter methylation in peripheral blood of TGCT patients can be associated with testicular cancer risk. Materials and methods Following a meta-analysis, we performed a cohort study including 32 testicular cancer patients and 32 healthy controls. Promoter methylation of the RASSF1A and O6-methylguanine-DNA-methyltransferase (MGMT) genes was analyzed using bisulfite pyrosequencing of DNA from peripheral blood. Results Meta-analysis showed an odds ratio (OR) of 7.69 for RASSF1A promoter methylation as a risk factor for TGCT. Cohort study found altered methylation of the RASSF1A promoter in blood of TGCT patients. Methylation was higher in TGCT patients before BEP chemotherapy. Conclusion The meta-analysis indicates a role of the RASSF1A promoter hypermethylation from peripheral blood in TCGT. We confirmed that finding in our cohort study, which represents the first report of changed RASSF1A promoter methylation in peripheral blood TGCT.

Background: The RAS association domain family protein 1a (RASSF1A) is a prominent tumor suppressor gene showing altered promoter methylation in testicular germ cell tumors (TGCT). RASSF1A promoter hypermethylation might represent an early event in TGCT tumorigenesis. We investigated whether the RASSF1A promoter methylation in peripheral blood of TGCT patients can be associated with testicular cancer risk. Materials and Methods: Following a meta-analysis, we performed a cohort study including 32 testicular cancer patients and 32 healthy controls. Promoter methylation of the RASSF1A and O6-methylguanine-DNA-methyltransferase (MGMT) genes was analyzed using bisulfite pyrosequencing of DNA from peripheral blood. Results: Meta-analysis showed an odds ratio (OR) of 7.69 for RASSF1A promoter methylation as a risk factor for TGCT. Cohort study found altered methylation of the RASSF1A promoter in blood of TGCT patients. Methylation was higher in TGCT patients before BEP chemotherapy. Conclusion: The meta-analysis indicates a role of the RASSF1A promoter hypermethylation from peripheral blood in TCGT. We confirmed that finding in our cohort study, which represents the first report of changed RASSF1A promoter methylation in peripheral blood TGCT.

Haematological and blood biochemical variables are key elements for routine assessment of animal health status, with implications in animal production and food safety. However, only reliable reference intervals of haematological and biochemical blood variables can provide the basis for accurate interpretation of the obtained blood results. Haematological and biochemical blood values critically dependent on living conditions, region, husbandry, rearing location, and diet. The aim of this study was to determine the reference intervals for selected important and informative haematological and blood biochemical variables for females of the Croatian indigenous sheep breed, Lika pramenka. We used several advanced statistical methods and assessed their applicability for determination of relevant reference intervals under conditions specific to indigenous sheep breeding. The results show that the reference intervals from the available literature are orientational but not adequate for the specific challenges encountered in raising indigenous breeds in their natural habitat. This was illustrated by the large number of clinically healthy animals with blood variable values falling outside the reference intervals given in the sources available previously., Hematološki i biokemijski krvni pokazatelji ključan su element pri rutinskom procjenjivanju zdravstvenog statusa životinja, produktivnosti životinja te sigurnosti hrane. Međutim, samo pouzdani referentni intervali hematoloških i biokemijskih pokazatelja u krvi mogu pružiti osnovu za precizno tumačenje dobivenih rezultata. Hematološke i biokemijske vrijednosti u krvi uvelike ovise o uvjetima držanja, regiji u kojoj se životinje drže, načinu uzgoja, mjestu uzgoja te hranidbi životinja. Cilj ovog istraživanja bio je utvrditi pouzdane referentne intervale za odabrane važne i informativne hematološke i biokemijske pokazatelje u krvi ženskih životinja hrvatske izvorne pasmine ličke pramenke. U istraživanju su korištene napredne statističke metode te je procijenjena njihova primjenjivost za određivanje relevantnih referentnih intervala u specifičnim uvjetima ovčarstva izvornim pasminama. Rezultati pokazuju da su referentni intervali iz dostupne literature orijentacijski, ali ne i dovoljni pri specifičnim izazovima u uzgoju izvornih pasmina u njihovom prirodnom staništu. To je ilustrirano velikim brojem klinički zdravih životinja čije se vrijednosti krvnih pokazatelja nalaze izvan referentnih intervala danim u prijašnjim istraživanjima.

Changes in N-glycosylation of plasma proteins are observed in many types of cancer, nevertheless, few studies suggest the exact mechanism involved in aberrant protein glycosylation. Here we studied the impact of DNA methylation on the N-glycome in the secretome of the HepG2 cell line derived from hepatocellular carcinoma (HCC). Since the majority of plasma glycoproteins originate from the liver, the HepG2 cells represent a good model for glycosylation changes in HCC that are detectable in blood, which is an easily accessible analytic material in a clinical setting. Two different concentrations of 5-aza-2′-deoxycytidine (5-aza-2dC) differentially affected global genome methylation and induced different glycan changes. Around twenty percent of 84 glyco-genes analysed changed expression level after the 5-aza-2dC treatment as a result of global genome hypomethylation. A correlation study between the changes in glyco-gene expression and the HepG2 glycosylation profile suggests that the MGAT3 gene might be responsible for the glycan changes consistently induced by both doses of 5-aza-2dC. Core-fucosylated tetra-antennary structures were decreased in quantity likely as a result of hypomethylated MGAT3 gene promoter followed by increased expression of this gene.

Purpose: Alternative glycosylation has significant structural and functional consequences on IgG and consequently also on cancer immunosurveillance. Because of technological limitations, the effects of highly heritable individual variations and the differences in the dynamics of changes in IgG glycosylation on colorectal cancer were never investigated before. Experimental Design: Using recently developed high-throughput UPLC technology for IgG glycosylation analysis, we analyzed IgG glycome composition in 760 patients with colorectal cancer and 538 matching controls. Effects of surgery were evaluated in 28 patients sampled before and three times after surgery. A predictive model was built using regularized logistic regression and evaluated using a 10-cross validation procedure. Furthermore, IgG glycome composition was analyzed in 39 plasma samples collected before initial diagnosis of colorectal cancer. Results: We have found that colorectal cancer associates with decrease in IgG galactosylation, IgG sialylation and increase in core-fucosylation of neutral glycans with concurrent decrease of core-fucosylation of sialylated glycans. Although a model based on age and sex did not show discriminative power (AUC = 0.499), the addition of glycan variables into the model considerably increased the discriminative power of the model (AUC = 0.755). However, none of these differences were significant in the small set of samples collected before the initial diagnosis. Conclusions: Considering the functional relevance of IgG glycosylation for both tumor immunosurveillance and clinical efficacy of therapy with mAbs, individual variation in IgG glycosylation may turn out to be important for prediction of disease course or the choice of therapy, thus warranting further, more detailed studies of IgG glycosylation in colorectal cancer. Clin Cancer Res; 22(12); 3078–86. ©2016 AACR.

Epigenetic alterations may provide important insights into gene-environment interaction in inflammatory bowel disease (IBD). Here we observe epigenome-wide DNA methylation differences in 240 newly-diagnosed IBD cases and 190 controls. These include 439 differentially methylated positions (DMPs) and 5 differentially methylated regions (DMRs), which we study in detail using whole genome bisulphite sequencing. We replicate the top DMP (RPS6KA2) and DMRs (VMP1, ITGB2 and TXK) in an independent cohort. Using paired genetic and epigenetic data, we delineate methylation quantitative trait loci; VMP1/microRNA-21 methylation associates with two polymorphisms in linkage disequilibrium with a known IBD susceptibility variant. Separated cell data shows that IBD-associated hypermethylation within the TXK promoter region negatively correlates with gene expression in whole-blood and CD8+ T cells, but not other cell types. Thus, site-specific DNA methylation changes in IBD relate to underlying genotype and associate with cell-specific alteration in gene expression., Epigenetic perturbations may be an important factor in diseases where both genes and environment play a role. Here, Ventham and colleagues show that DNA methylation changes in inflammatory bowel disease are related to the underlying genotype, and are associated with cell-specific changes to gene expression.

Background Most eukaryotic proteins are modified by covalent addition of glycan molecules that considerably influence their function. Aberrant glycosylation is profoundly involved in malignant transformation, tumor progression and metastasis. Some glycan structures are tumor-specific and reflect disturbed glycan biosynthesis pathways. Methods We analyzed DNA methylation and expression of 86 glyco-genes in melanoma, hepatocellular, breast and cervical cancers using data from publicly available databases. We also analyzed methylation datasets without the available matching expression data for glyco-genes in lung cancer, and progression of melanoma into lymph node and brain metastases. Results Ten glyco-genes (GALNT3, GALNT6, GALNT7, GALNT14, MGAT3, MAN1A1, MAN1C1, ST3GAL2, ST6GAL1, ST8SIA3) showing changes in both methylation and expression in the same type of cancer belong to GalNAc transferases, GlcNAc transferases, mannosidases and sialyltransferases, which is in line with changes in glycan structures already reported in the same type of tumors. Some of those genes were additionally identified as potentially valuable for disease prognosis. The MGAT5B gene, so far identified as specifically expressed in brain, emerged as a novel candidate gene that is epigenetically dysregulated in different cancers other than brain cancer. We also report for the first time aberrant expression of the GALNT and MAN genes in cancer by aberrant promoter methylation. Conclusions Aberrant expression of glyco-genes due to aberrant promoter methylation could be a way leading to characteristic glycosylation profiles commonly described in cancer. General significance Methylation status in promoters of candidate glyco-genes might serve as prognostic markers for specific tumors and point to potential novel targets for epigenetic drugs. This article is part of a Special Issue entitled "Glycans in personalised medicine" Guest Editor: Professor Gordan Lauc.

Genome editing tools, such as TALEN (transcription activator-like effector nuclease) or CRISPR-Cas9 (CRISPR-associated protein-9 nuclease) systems, enable functional studies by targeted gene knockout. They introduce double-stranded breaks (DSBs) into a DNA molecule in a sequence-specific manner, thereby stimulating the error-prone non-homologous end joining repair mechanism, leading to probable gene inactivation when the coding sequence is targeted. Vectors for expression of TALEN and Cas9-based constructs targeting the human IL6ST and HNF1A genes were assembled and tested for their ability to introduce DSBs when transfected into cultured cells using the luciferase assay. The Cas9-based construct targeting the IL6ST gene was shown to be active, while the two TALEN-based constructs did not introduce DSBs above background level. Both the TALEN and the CRISPR-Cas9 constructs targeting the HNF1A gene were found to be active, with the TALEN showing higher activity in a dose-dependent manner. The constructed genome-editing tools can be used for functional analysis of the putative role of HNF1A and IL6ST genes in IgG glycosylation, as shown previously by genome wide association studies. This work is licensed under a Creative Commons Attribution 4.0 International License.

Background: Since testicular cancer shows different disease progress depending on etiology and the neoplastic tissue profile, with epigenetic changes being a likely underlying mechanism, we investigated CpG methylation of MGMT and RASSF1, which had been identified as promising candidate biomarkers for disease progression, chemotherapy success and prognosis. Methods: Genomic DNA was isolated from blood of 32 healthy individuals and 32 patients with testicular cancer before and after the chemotherapy: etoposide, cisplatin, bleomycin (BEP). Eighteen samples were seminomas and 14 were nonseminomas. For quantitative measurement of DNA methylation levels in MGMT and RASSF1A, bisulfite-converted DNA was amplified by PCR and then sequenced using a pyrosequencing technology. Results: Methylation levels were not significantly different for MGMT before and after chemotherapy ; however, methylation of the tested CpG sites in RASSF1 was significantly higher in patients before treatment compared with controls at 5 of 9 sites, two sites remaining significant even after the Bonferroni correction. Methylation of RASSF1 approached that of the controls after treatment. In addition, we found indications that additional stratification by histopathological profile might add value to MGMT and RASSF1 methylation as a prognostic marker. The methylation of LINE1 elements was similar in patients before and after chemotherapy. Conclusions: Increasing statistical power by studying larger cohorts has a potential to further confirm RASSF1 methylation as a chemotherapy success biomarker, while additional stratification by histopahological characteristics could give further insight leading to more accurate prognosis using both RASSF1 and MGMT, thereby enabling better risk- adapted management of testicular cancer.