Are there new biomarkers of the gastroduodenal microbiota useful in the diagnosis of coeliac disease in children? A pilot study.

The changing of microbiome could precede the development of coeliac disease (CeD). We compared the bacterial profile of microbiota of tissues collected simultaneously from the stomach and duodenum in newly diagnosed patients with CeD. Biopsies were collected from 60 children and adolescents aged 2-18 years: (1) 40 patients with CeD; (2) 20 children as control group. The evaluation of the bacterial microbiota was carried out by sequencing the V3-V4 regions of the 16S rRNA subunit, using next-generation sequencing (NGS). The composition of bacterial microbiota was correlated with clinical and blood parameters. The beta diversity analysis revealed a significant dissimilarity in the gastric samples between the CeD and control group (Bray-Curtis index, P = 0.008, and weighted UniFrac distance, P = 0.024). At L2 (phylum level), Campylobacterota was only present in the stomach of the CeD group. A comparison of the abundance of bacteria between the stomach and duodenum showed significant differences in 10 OTUs (operational taxonomic units) in the control and 9 OTUs in the CeD group at L6 (genus) and in 8 OTUs and in 6 OTUs, respectively, at L7 (species). A significant correlation was observed between the genus Novosphingobium in stomach of CeD group and possession of the DQ2.5 and DQ 8 allele, and in the duodenum - between the DQ 8 allele and the species Blautia wexlerae. Significant differences in selected, little-known genera of bacteria suggest their potential role as new biomarkers in the development of CeD. To fully understand the mechanism of CeD development in genetically predisposed individuals, it is necessary to take into account not only the abundance of a given genus or species of bacteria, but also the anatomical location of its occurrence.

Transcriptomic profiling of mare endometrium at different stages of endometrosis.

In the current study, transcriptome profiles of mare endometrium, classified into categories I, IIA, and IIB according to Kenney and Doig, were compared using RNA sequencing, analyzed, and functionally annotated using in silico analysis. In the mild stage (IIA) of endometrosis compared to category I endometrium, differentially expressed genes (DEGs) were annotated to inflammation, abnormal metabolism, wound healing, and quantity of connective tissue. In the moderate stage (IIB) of endometrosis compared to category I endometrium, DEGs were annotated to inflammation, fibrosis, cellular homeostasis, mitochondrial dysfunction, and pregnancy disorders. Ingenuity pathway analysis (IPA) identified cytokines such as transforming growth factor (TGF)-ß1, interleukin (IL)-4, IL-13, and IL-17 as upstream regulators of DEGs associated with cellular homeostasis, metabolism, and fibrosis signaling pathways. In vitro studies showed the effect of these cytokines on DEGs such as ADAMTS1, -4, -5, -9, and HK2 in endometrial fibroblasts at different stages of endometrosis. The effect of cytokines on ADAMTS members' gene transcription in fibroblasts differs according to the severity of endometrosis. The identified transcriptomic changes associated with endometrosis suggest that inflammation and metabolic changes are features of mild and moderate stages of endometrosis. The changes of ADAMTS-1, -4, -5, -9, in fibrotic endometrium as well as in endometrial fibroblast in response to TGF-ß1, IL-4, IL-13, and IL-17 suggest the important role of these factors in the development of endometrosis.

Next-Generation Sequencing analysis discloses genes implicated in equine endometrosis that may lead to tumorigenesis.

Endometrosis is a periglandular fibrosis associated with dysfunction of affected glandular epithelial cells that is the most common cause of reduced fertility in mares, although it is not fully understood. The etiology of the disease is still partially unknown. This study focuses on understanding the genetic mechanisms potentially underlying endometrosis in mares using the Next Generation Sequencing (NGS) technique. Endometrial samples, used in the study, were obtained in the anestrus phase both from healthy mares and those diagnosed with endometrosis. The NGS data were analyzed for gene involvement in biological processes and pathways (e.g. STAR, KOBAS-I, STRING, and ClustVis software). Bioinformatic analysis revealed differential expression of 55 transcripts. In tissues with endometrosis, most genes displayed upregulated expression. The protein-protein interaction analysis disclosed a substantial transcript network including transcripts related to metabolism e.g. sulfur metabolism (SELENBP1), ovarian steroidogenesis, steroid hormone biosynthesis, and chemical carcinogenesis (CYP1B1), COXs (COX4I1, COX3, UQCRFS1) as well as transcripts related to immune response e.g. MMP7, JCHAIN, PIGR, CALR, B2M, FCGRT. Interestingly, the latter has been previously linked with various pathologies including cancers in the female reproductive system. In conclusion, this study evaluated genes that are not directly impacted by sex hormone feedback, but that create a metabolic and immune environment in tissues, thus influencing fertility and pregnancy in mares with endometrosis. Moreover, some of the identified genes may be implicated in tumorigenesis of endometrial lesions. These data may be useful as a starting point in further research, such as the development of targeted strategies for rapid diagnosis and/or prevention of this pathology based on gene and protein-protein interactions.

Mouse testicular transcriptome after modulation of non-canonical oestrogen receptor activity.

The aims of this study were to shed light on the role of G-protein-coupled membrane oestrogen receptor (GPER) and oestrogen-related receptor (ERR) in mouse testis function at the gene expression level, as well as the involvement of GPER and ERR in cellular and molecular processes. Male mice were injected (50µg kg-1,s.c.) with the GPER antagonist G-15, the ERRα inverse agonist XCT790 or the ERRß/ERRγ agonist DY131. Next-generation sequencing (RNA-Seq) was used to evaluate gene expression. Bioinformatic analysis of read abundance revealed that 50, 86 and 171 transcripts were differentially expressed in the G-15-, XCT790- and DY131-treated groups respectively compared with the control group. Annotated genes and their protein products were categorised regarding their associated biological processes and molecular functions. In the XCT790-treated group, genes involved in immunological processes were upregulated. In the DY131-treated group, genes with increased expression were primarily engaged in protein modification (protein folding and small protein conjugation). In addition, the expression of genes recognised as oncogenes, such as BMI1 proto-oncogene, polycomb ring finger (Bmi1) and nucleophosphin 1 (Npm1), was significantly increased in all experimental groups. This study provides detailed information regarding the genetic changes in the testicular transcriptome of the mouse in response to modulation of non-canonical oestrogen receptor activity.

Methylation Marks of Blood Leukocytes of Native Hucul Mares Differentiated in Age.

Horses are one of the longest-living species of farm animals. Advanced age is often associated with a decrease in body condition, dysfunction of immune system, and late-onset disorders. Due to this, the search for new solutions in the prevention and treatment of pathological conditions of the advanced age of horses is desirable. That is why the identification of aging-related changes in the horse genome is interesting in this respect. In the recent years, the research on aging includes studies of age-related epigenetic effects observed on the DNA methylation level. We applied reduced representation bisulfite sequencing (RRBS) to uncover a range of age DMR sites in genomes of blood leukocytes derived from juvenile and aged horses of native Hucul breed. Genes colocated with age-related differentially methylated regions (age DMRs) are the members of pathways involved in cellular signal transduction, immune response, neurogenesis, differentiation, development, and cancer progression. A positive correlation was found between methylation states and gene expression in particular loci from our data set. Some of described age DMR-linked genes were also reported elsewhere. Obtained results contribute to the knowledge about the molecular basis of aging of equine blood cells.

Characterisation of genome-wide structural aberrations in canine mammary tumours using single nucleotide polymorphism (SNP) genotyping assay.

Characterisation of copy number variation (CNV) and loss of heterozygosity (LOH) has pro- vided evidence for the relationship of this type of genetic variation with the occurrence of a broad spectrum of diseases, including cancer lesions. The role of CNVs and germinal or somatic LOHs in canine mammary tumours is still unknown. Therefore, the aim of this study was to identify CNVs and LOHs in canine mammary tumours. Forty-eight samples obtained from normal (n=24) and tumour (n=24) tissues of dogs were analysed. In the study, we used CanineHD BeadChip assay (Illumina) and OncoSNP software to identify copy number alternations in genomes of dif- ferent dog breeds and in different mammary cancer types occurring in this species. The analyses revealed that, in the case of CNV, the amplification-type variants were longer and more frequent than deletions. Based on the analysis of the frequency of different types of aberrations in the in- dividual parts of the genome, regions that are particularly susceptible to structural aberrations were indicated. The fraction of genes identified within these regions was associated with major processes of neoplastic transformation. Association analysis of such traits as tumour grading as well as the size and age of dogs demonstrated that structural aberrations were more frequent in dogs diagnosed with tumour malignancy grade II and III, in dogs with a larger body size, and in large dogs aged 7-8. The promising results of these pioneering investigations prompt continuation thereof to analyse other types of cancer.

DNA methylation patterns of the S100A14, POU2F3 and SFN genes in equine sarcoid tissues.

Genetic and epigenetic alterations in the equine sarcoid, a locally invasive skin tumour of equids, are still poorly characterized. Numerous studies have provided reliable evidence for the relationship between the development of cancer and the loss of function of a number of tumour suppressor genes. In the present study, we assessed methylation levels in the promoter region of SFN, S100A14 and POU2F3 genes in sarcoid samples to clarify whether DNA methylation may be associated with previously identified changes in the expression level of these genes during the course of tumour progression. Using bisulfite sequencing and clone sequencing, we detected that lesional samples had a significantly higher rate of DNA methylation in the analyzed S100A14A region than the corresponding normal skin tissue. A frequent methylation of the SFN and POU2F3 promoter sequences were observed in both the tumour samples and the control skin tissues. Further studies are needed to evaluate the role of aberrant methylation in sarcoid progression and to understand the mechanisms involved in reduced expression of SFN, S100A14 and POU2F3 genes in the lesional tissues.

Comparative analysis of DNA methylation patterns of equine sarcoid and healthy skin samples.

OBJECTIVE: In this study, for the first time we report the genome-wide DNA methylation profile of skin tumour in horses and describe differentially methylated genomic regions (DMRs) with respect to healthy skin. MATERIALS & METHODS: The comparative analysis of DNA methylation patterns detected using Reduced Representation Bisulfite Sequencing (RRBS) technique, allowed identification of 136 regions showing differential methylation between sarcoid and normal skin tissue. RESULTS: Most of the identified DMRs were short fragments, less than 1 kb in size, located in the intergenic regions. Among identified DMRs there were also regions located within genes directly or indirectly related with oncogenesis. We additionally validated 9 CpG sites showing hypomethylation and 9 CpG sites that were hypermethylated in lesional sample, confirming the identified changes in the DNA methylation. CONCLUSION: Knowledge on the changes taking place in the process of DNA methylation may provide a basis for the development of new alternative diagnostic or therapeutic approaches to equine sarcoids.

Transcriptome analysis of equine sarcoids.

Equine sarcoids are the most commonly detected skin tumours in Equidae. In the present research, a comparative transcriptomic analysis was performed which aimed at looking inside a tumour biology and identification of the expression profile as a potential source of cancer specific genes useful as biomarkers. We have used Horse Gene Expression Microarray data from matched equine sarcoids and tumour-distant skin samples. In total, 901 significantly differentially expressed genes (DEGs) between lesional and healthy skin samples have been identified (fold change ≥ 2; P < 0.05). The large subset of DEGs, with decreased expression, was associated with a suppression of malignant transformation, whereas several overexpressed genes were involved in the processes associated with growth and progression of a tumour or immune system activity. Our results, as a first to date, showed comprehensive transcriptome analysis of skin tumour in horses and pinpointed significant pathways and genes related with oncogenesis processes.

Relevance of Molecular Changes in the ND4 Gene in German Shepherd Dog Tumours.

The aim of the study was to identify polymorphisms and mutations in the mitochondrial ND4 gene and to analyse the associations between the occurrence of molecular changes in mtDNA and phenotypic traits in tumours in German Shepherd dogs. Fifty samples obtained from blood and tumour tissues of German Shepherd dogs with diagnosed tumours were analysed. DNA extraction, amplification, and sequencing of the mtDNA ND4 gene, and bioinformatics, statistical, and in silico protein coding SNP analyses were performed. ND4 mutations and/or polymorphisms were noted in eleven nucleotide positions in nearly half of the examined dogs. All the changes were substitution mutations. A majority of the changes identified were homoplasmic. In one dog with osteosarcoma, blood heteroplasmy was detected. In two positions of the ND4 gene, presence of non-synonymous mutations leading to amino acid changes in the ND4 protein was reported. Analyses carried out to determine the deleterious effect of mutations indicated an almost 97 and 62% probability that a single amino acid substitution (p.G239V and p.I401T, respectively) in the protein has a negative impact on its function. The results of statistical analyses indicate a significant association between the occurrence of mutations in three loci of the ND4 gene and the location of tumours. The mutations identified may be a result of cell adaptation to the changes in the environment occurring during carcinogenesis. The high frequency of mutations in the tumours may indicate genetic instability of mtDNA, which may also play a role in carcinogenesis.

The use of runs of homozygosity for estimation of recent inbreeding in Holstein cattle.

Controlling inbreeding in livestock populations is of great importance because excess relatedness among animals leads to a rapid loss of genetic variation and to adverse phenotypical effects associated with an inbreeding depression. Recent advances in genotyping technology have made it possible to study inbreeding at a molecular level by the analysis of genome-wide single nucleotide polymorphism panels. In this study, we used BovineSNP50 assay (Illumina) to estimate genomic inbreeding coefficient in 298 Holstein cattle by the analysis of the genome portion in runs of homozygosity (FROH) or using genomic relationship matrix (FGRM), and compared this data with conventional pedigree-based inbreeding coefficients (FPED). Weak or moderate Spearman's rank correlations were observed between FROH and FPED which depended on the ROH length categories used for calculations and inclusion of animals with different number of complete generations registered in pedigrees. The highest correlations were observed when using ROH with lengths over 8 Mb (0.334). The correlations tended to increase as pedigree depth increased, and were the highest for animals with seven complete generations of pedigree data. FGRM correlated poorly with pedigree-based estimates, which suggests that ROH-based inbreeding coefficients better reflect recent relatedness among animals.

Genome-wide RNA-Seq analysis of breast muscles of two broiler chicken groups differing in shear force.

In this study, a whole transcriptome analysis of breast muscles was conducted in broiler chicken groups differing in shear force. Shear force is a determinant of tenderness, which in turn is one of the most important parameters of meat quality in chickens. In our analysis, a total of 11,560 transcripts and 9824 genes per sample were identified. In chickens with more tender meat, up-regulation of 19 genes and down-regulation of 49 genes was observed. The up-regulated gene group included the ASB2 gene, which is probably involved in the meat conversion process, as its product results in the degradation of filamins, proteins which form muscle fibres. In the down-regulated gene group, genes which play a role in lipogenesis (THRSP, PLIN1) and in collagen synthesis (P4HA3, LEPREL4, PCOLCE2, COL16A1, COL20A1, VWA1) were detected. Their presence suggests the involvement of the extracellular matrix in the determination of meat tenderness. Thus, our study identified a pool of genes that may participate in the tenderisation process in broiler chickens.

Identification of genome-wide selection signatures in the Limousin beef cattle breed.

The study is aimed at identifying selection footprints within the genome of Limousin cattle. With the use of Extended Haplotype Homozygosity test, supplemented with correction for variation in recombination rates across the genome, we created map of selection footprints and detected 173 significant (p < 0.01) core haplotypes being potentially under positive selection. Within these regions, a number of candidate genes associated inter alia with skeletal muscle growth (GDF15, BMP7, BMP4 and TGFB3) or postmortem proteolysis and meat maturation (CAPN1 and CAPN5) were annotated. Noticeable clusters of selection footprints were detected on chromosomes 1, 4, 8 and 14, which are known to carry several quantitative trait loci for growth traits and meat quality. The study provides information about the genes and metabolic pathways potentially modified under the influence of directional selection, aimed at improving beef production characteristics in Limousin cattle.

Genome-wide characteristics of copy number variation in Polish Holstein and Polish Red cattle using SNP genotyping assay.

Copy number variation (CNV), which results from deletions or amplifications of large fragments of genomic DNA, is widespread in mammalian genomes and apart from its potential pathogenic effect it is considered as a source of natural genetic diversity. In cattle populations, this kind of genetic variability remains still insufficiently elucidated and studies focusing on the detection of new structural genomic variants in different cattle populations may contribute to a better understanding of cattle breeds' diversity and genetic basis of production traits. In this study, by using BovineSNP50 assay and cnvPartition algorithm we identified CNVs in two different cattle breeds: Holstein (859 animals) and Polish Red (301). In Holstein cattle we found 648 CNVs which could be reduced to 91 non-redundant variable genomic regions (CNVRs) covering in total 168.6 Mb of the genomic sequence. In Polish Red cattle we detected 62 CNVs, localized in 37 variable regions encompassing 22.3 Mb of the sequence, corresponding to 0.89 % of the autosomal genome. Within the regions we identified 1,192 unique RefSeq genes which are engaged in a variety of biological processes. High concordance of the regions' distribution was found between the studied breeds, however copy number variants seemed to be more common in Holstein cattle. About 26 % of the regions described in this study could be classified as newly identified. The results of this study will broaden the knowledge of CNVs in genomes of cattle of different breeds and will provide foundations for further research aiming to identify a relationship between this type of genetic variation and phenotypic traits.

Epigenetic structure and the role of polymorphism in the shaping of DNA methylation patterns of equine OAS1 locus.

DNA methylation patterns and their relation with genetic polymorphisms were determined in the equine OAS1 locus. Genetic variants of OAS1 were previously found to be associated with susceptibility to West Nile virus infections in horses. The subject of the study were white blood cells of 13 juvenile and 13 old horses from AA and HC breed and a set of solid tissues from a single adult horse. The aim was to determine the degree of variation of CpG methylation profiles with concern for tissue type, horse breed and age. Results of direct BSPCR and cloned BSPCR sequencing revealed that all of determined CpG islands (CGIs) were hypermethylated in exception to CGI covering OAS1 promoter and exon 1. One of intragenic CGIs displayed variability of methylation patterns across eight tissue types. The variability of particular sub-types of white blood cells between AA and HC horses were considered as the possible cause of interbreed differences of methylation levels. Comparison of sequence variability between converted and unconverted DNAs of both horse breeds showed polymorphisms of CpG sites to be the source of monoallelic methylation in exception to the polymorphic CpGs located in the OAS1 promoter. Two of them are new polymorphic variants in the OAS1 promoter region. Application of methylation data in conjunction with genetic variation detected at the OAS1 locus might be useful to deepen the knowledge about mechanisms underlying immunity to viral infections in the horse.

Identification of carriers of the mutation causing coagulation factor XI deficiency in Polish Holstein-Friesian cattle.

Factor XI (FXI) deficiency is a hereditary coagulation disorder observed in various mammalian species. The molecular basis of coagulopathy has been recognized in Holstein cattle as a 76-bp insertion in the coding region of the FXI gene. Because the disorder seems to have an impact on reproductive traits and udder health in cattle, we tested 103 randomly selected cows, 28 cows with repeat breeding, and 9 cows with recurrent mastitis for the presence of an abnormal FXI allele. Three related cows were diagnosed as carriers.