Bioinformatic analysis of endometrial miRNA expression profile at day 26-28 of pregnancy in the mare.

The establishment of the fetomaternal interface depends on precisely regulated communication between the conceptus and the uterine environment. Recent evidence suggests that microRNAs (miRNAs) may play an important role in embryo-maternal dialogue. This study aimed to determine the expression profile of endometrial miRNAs during days 26-28 of equine pregnancy. Additionally, the study aimed to predict target genes for differentially expressed miRNAs (DEmiRs) and their potential role in embryo attachment, adhesion, and implantation. Using next-generation sequencing, we identified 81 DEmiRs between equine endometrium during the pre-attachment period of pregnancy (day 26-28) and endometrium during the mid-luteal phase of the estrous cycle (day 10-12). The identified DEmiRs appear to have a significant role in regulating the expression of genes that influence cell fate and properties, as well as endometrial receptivity formation. These miRNAs include eca-miR-21, eca-miR-126-3p, eca-miR-145, eca-miR-451, eca-miR-491-5p, members of the miR-200 family, and the miRNA-17-92 cluster. The target genes predicted for the identified DEmiRs are associated with ion channel activity and sphingolipid metabolism. Furthermore, it was noted that the expression of mucin 1 and leukemia inhibitory factor, genes potentially regulated by the identified DEmiRs, was up-regulated at day 26-28 of pregnancy. This suggests that miRNAs may play a role in regulating specific genes to create a favorable uterine environment that is necessary for proper attachment, adhesion, and implantation of the embryo in mares.

The potential role of miRNAs and regulation of their expression in the development of mare endometrial fibrosis.

Mare endometrial fibrosis (endometrosis), is one of the main causes of equine infertility. Despite the high prevalence, both ethology, pathogenesis and the nature of its progression remain poorly understood. Recent studies have shown that microRNAs (miRNAs) are important regulators in multiple cellular processes and functions under physiological and pathological circumstances. In this article, we reported changes in miRNA expression at different stages of endometrosis and the effect of transforming growth factor (TGF)-ß1 on the expression of the most dysregulated miRNAs. We identified 1, 26, and 5 differentially expressed miRNAs (DEmiRs), in categories IIA (mild fibrosis), IIB (moderate fibrosis), and III (severe fibrosis) groups compared to category I (no fibrosis) endometria group, respectively (Padjusted < 0.05, log2FC ≥ 1.0/log2FC ≤ - 1.0). This study indicated the potential involvement of miRNAs in the regulation of the process associated to the development and progression of endometrosis. The functional enrichment analysis revealed, that DEmiRs target genes involved in the mitogen-activated protein kinases, Hippo, and phosphoinositide-3-kinase (PI3K)-Akt signalling pathways, focal adhesion, and extracellular matrix-receptor interaction. Moreover, we demonstrated that the most potent profibrotic cytokine-TGF-ß1-downregulated novel-eca-miR-42 (P < 0.05) expression in fibroblasts derived from endometria at early-stage endometrosis (category IIA).

Transcriptome and methylome sequencing reveals altered long non-coding RNA genes expression and their aberrant DNA methylation in equine sarcoids.

Recent publications confirmed that long non-coding RNAs (lncRNAs) perform an essential function in gene-specific transcription regulation. Nevertheless, despite its important role, lncRNA has not yet been described in equine sarcoids, the skin neoplasia of horses. Therefore, the aim of this study is to deepen the knowledge about lncRNA expression in the pathogenesis of equine sarcoids and provide new insight into the regulatory function of lncRNA in the bovine papillomavirus-dependent neoplasia of horse dermal tissues. RNA sequencing (RNA-seq) data from 12 equine sarcoid samples and the corresponding controls were reanalyzed in this study. A total of 3396 differentially expressed (DE) lncRNAs and 128 DElncRNA-DE genes (DEGs) pairs were identified. Differentially expressed lncRNAs predicted target genes were enriched in pathways associated with inter alia the extracellular matrix disassembly and cancer pathways. Furthermore, methylation data from the same samples were integrated into the analysis, and 12 DElncRNAs were described as potentially disturbed by aberrant methylation. In conclusion, this study presents novel data about lncRNA's role in the pathogenesis of equine sarcoids.

Methylome and transcriptome data integration reveals aberrantly regulated genes in equine sarcoids.

DNA methylation is a key mechanism in transcription regulation, and aberrant methylation is a common and important mechanism in tumor initiation, maintenance, and progression. To find genes that are aberrantly regulated by altered methylation in horse sarcoids, we used reduced representation bisulfite sequencing (RRBS) accompanied by RNA sequencing (RNA-Seq) for methylome (whole genome DNA methylation sequencing) and transcriptome profiling, respectively. We found that the DNA methylation level was generally lower in lesion samples than in controls. In the analyzed samples, a total of 14,692 differentially methylated sites (DMSs) in the context of CpG (where cytosine and guanine are separated by a phosphate), and 11,712 differentially expressed genes (DEGs) were identified. The integration of the methylome and transcriptome data suggests that aberrant DNA methylation may be involved in the deregulation of expression of the 493 genes in equine sarcoid. Furthermore, enrichment analysis of the genes demonstrated the activation of multiple molecular pathways related to extracellular matrix (ECM), oxidative phosphorylation (OXPHOS), immune response, and disease processes that can be related to tumor progression. The results provide further insight into the epigenetic alterations in equine sarcoids and provide a valuable resource for follow-up studies to identify biomarkers for predicting susceptibility to this common condition in horses.

MicroRNA gene methylation landscape in pediatric B-cell precursor acute lymphoblastic leukemia.

BACKGROUND: Aberrant DNA methylation is an important mechanism by which the normal patterns of microRNA expression are disrupted in human cancers including B-cell precursor acute lymphoblastic leukemia (BCP ALL), the most common pediatric malignancy. OBJECTIVES: To characterize the methylation profile landscape of microRNA genes in BCP ALL patients. MATERIAL AND METHODS: We employed Infinium® MethylationEPIC BeadChip Arrays to measure the methylation of microRNA genes from bone marrow samples of children with BCP ALL (n = 38) and controls without neoplasms (n = 4). RESULTS: This analysis revealed differential methylation of the microRNA genes in the pediatric BCP ALL when compared to the control. A subcluster amongst BCP ALL patients with TCF3-PBX1 genetic subtype was also observed. No other differences were observed in association with age, gender or risk group. Several interesting leukemia-related phenotypes are enriched by the genes with hyperand hypomethylated sites located in promoters as well as gene bodies. The top 3 miRNA genes, promoters of which were the most statistically significantly hypermethylated in BCP ALL were MIR1273G, MIR1304 and MIR663, and the top 3 hypomethylated were MIR4442, MIR155 and MIR3909. CONCLUSIONS: In this study, a different microRNA genes methylation landscape was shown in pediatric BCP ALL compared to children without neoplasms. A visible subcluster among BCP ALL samples consisted of individuals with TCF3-PBX1 genetic subtype. No other differences were observed in association with age, gender or risk group. Several interesting leukemia-connected phenotypes were found, associated with genes with hyperand hypomethylated sites located on promoters as well as gene bodies.

Peroxisome Proliferator-Activated Receptor γ, but Not α or G-Protein Coupled Estrogen Receptor Drives Functioning of Postnatal Boar Testis-Next Generation Sequencing Analysis.

Porcine tissue gene expression is highly similar to the expression of homologous genes in humans. Based on this fact, the studies on porcine tissues can be employed to understand human physiology and to predict or treat diseases. Our prior studies clearly showed that there was a regulatory partnership of the peroxisome proliferator-activated receptor (PPAR) and the G-protein coupled membrane estrogen receptor (GPER) that relied upon the tumorigenesis of human and mouse testicular interstitial cells, as well as the PPAR-estrogen related receptor and GPER-xenoestrogen relationships which affected the functional status of immature boar testes. The main objective of this study was to identify the biological processes and signaling pathways governed by PPARα, PPARγ and GPER in the immature testes of seven-day-old boars after pharmacological receptor ligand treatment. Boar testicular tissues were cultured in an organotypic system with the respective PPARα, PPARγ or GPER antagonists. To evaluate the effect of the individual receptor deprivation in testicular tissue on global gene expression, Next Generation Sequencing was performed. Bioinformatic analysis revealed 382 transcripts with altered expression. While tissues treated with PPARα or GPER antagonists showed little significance in the enrichment analysis, the antagonists challenged with the PPARγ antagonist displayed significant alterations in biological processes such as: drug metabolism, adhesion and tubule development. Diverse disruption in the Notch signaling pathway was also observed. The findings of our study proposed that neither PPARα nor GPER, but PPARγ alone seemed to be the main player in the regulation of boar testes functioning during early the postnatal developmental window.

Transcriptome analysis of human Leydig cell tumours reveals potential mechanisms underlying its development.

Leydig cell tumours are the most common sex cord-stromal tumours. In the last years, apparent increased incidence is noted while aetiology of the tumour is still unknown. Therefore, here, we focused on the genetics of Leydig cell tumours using the next-generation sequencing. Leydig cell micronodules were revealed in patients with azoospermia who were qualified for testicular biopsy. Complete gene set of Leydig cell tumours was compared with transcriptome of healthy Leydig cells obtained from donors. Bioinformatic analysis of the obtained sequencing data revealed alterations in expression of 219 transcripts. We showed, for the first time, that a significant proportion of differentially expressed genes is directly involved in regulation of apoptotic process, which downregulation might be important to Leydig cell tumour development. Additionally, we found a significant upregulation of heat shock protein genes that might be a unique feature of Leydig cell tumours when compared to other tumour types. Our study offers fundamental transcriptomic data for future studies on human Leydig cell tumour that are crucial to determine its causes. Moreover, presented here the in-depth analysis and discussion of alterations observed in tumour transcriptome may be important for the diagnosis and therapy of this pathology.

Maternal atopy and offspring epigenome-wide methylation signature.

The increase in the prevalence of allergic diseases is believed to partially depend on environmental changes. DNA methylation is a major epigenetic mechanism, which is known to respond to environmental factors. A number of studies have revealed that patterns of DNA methylation may potentially predict allergic diseases.Here, we examined how maternal atopy is associated with methylation patterns in the cord blood of neonates.We conducted an epigenome-wide association study in a cohort of 96 mother-child pairs. Pregnant women aged not more than 35 years old, not currently smoking or exposed to environmental tobacco smoke, who did not report obesity before conception were considered eligible. They were further tested for atopy. Converted DNA from cord blood was analysed using Infinium MethylationEPIC; for statistical analysis, RnBeads software was applied. Gestational age and sex were included as covariates in the final analysis.83 DM sites were associated with maternal atopy. Within the top DM sites, there were CpG sites which mapped to genes SCD, ITM2C, NT5C3A and NPEPL1. Regional analysis revealed 25 tiling regions, 4 genes, 3 CpG islands and 5 gene promoters, (including PIGCP1, ADAM3A, ZSCAN12P1) associated with maternal atopy. Gene content analysis revealed pointwise enrichments in pathways related to purine-containing compound metabolism, the G1/S transition of the mitotic cell cycle, stem cell division and cellular glucose homoeostasis.These findings suggest that maternal atopy provides a unique intrauterine environment that may constitute the first environment in which exposure is associated with methylation patterns in newborn.

Alterations in the rainbow trout (Oncorhynchus mykiss) eggs exposed to ionizing radiation during induced androgenesis.

Ionizing radiation (IR) is applied to inactivate nuclear genome in the salmonid eggs to induce androgenetic development. However, it has been considered that doses of IR used to damage maternal chromosomes may also affect morphology of the eggs and decrease their developmental potential. Thus, the main goal of the present research was to assess alterations in the rainbow trout (Oncorhynchus mykiss) eggs caused by the high dose of IR administered during androgenesis. In the present research, rainbow trout eggs were irradiated with 350 Gy of X-rays, inseminated and exposed to the high hydrostatic pressure (HHP) shock to develop as androgenetic doubled haploids (DHs). The distribution of lipid droplets in the irradiated and non-irradiated rainbow trout eggs, survival rates and morphology of larvae from androgenetic and control groups were compared. It has been observed that non-irradiated and irradiated eggs exhibited altered distribution of lipid droplets. Most of the eggs before IR treatment displayed rather equal distribution of the oil droplets. In turn, majority of eggs studied after irradiation had coalesced lipid droplets, a pattern found in eggs with reduced quality. Incidences of abnormally developed larvae were more frequently observed among fish that hatched from the irradiated eggs. Observed changes suggest X-rays applied for the genetic inactivation of rainbow trout eggs may lead to decrease of their developmental competence.

The distinguishable DNA whole genome methylation profile of 2 cases of pediatric precursor B acute lymphoblastic leukaemia (BCP ALL) with prodromal, preleukemic phase: A case report.

RATIONALE: A prolonged, prodromal phase before definitive paediatric precursor B acute lymphoblastic leukaemia (BCP ALL) diagnosis is rarely observed. PATIENTS CONCERNS: In the first, the patient presented with an aplastic preleukemic phase, whilst the second presented with a rheumatic-like preliminary phase. DIAGNOSES: The case reports of two patients with BCP ALL with a prodromal phase lasting a few weeks are presented. INTERVENTIONS AND OUTCOMES: DNA whole genome profile methylation analysis of bone marrow cells obtained at diagnosis revealed a pattern of methylation that was readily distinguishable from both healthy and standard course BCP ALL bone marrow samples. LESSONS: The biological implication of this observation remains unclear, with many differentially methylated loci involved in many processes like neurogenesis, cell projection organization and adhesion along with leucocyte activation and apoptosis. The prevalence and clinical significance of these methylation changes is unknown but this data indicates that the epigenetic basis of BCP ALL with a prolonged, prodromal phase requires a more detailed assessment.

A comprehensive transcriptome analysis of skeletal muscles in two Polish pig breeds differing in fat and meat quality traits

Abstract Pork is the most popular meat in the world. Unfortunately, the selection pressure focused on high meat content led to a reduction in pork quality. The present study used RNA-seq technology to identify metabolic process genes related to pork quality traits and fat deposition. Differentially expressed genes (DEGs) were identified between pigs of Pulawska and Polish Landrace breeds for two the most important muscles (semimembranosus and longissimus dorsi). A total of 71 significant DEGs were reported: 15 for longissimus dorsi and 56 for semimembranosus muscles. The genes overexpressed in Pulawska pigs were involved in lipid metabolism (APOD, LXRA, LIPE, AP2B1, ENSSSCG00000028753 and OAS2) and proteolysis (CST6, CTSD, ISG15 and UCHL1). In Polish Landrace pigs, genes playing a role in biological adhesion (KIT, VCAN, HES1, SFRP2, CDH11, SSX2IP and PCDH17), actin cytoskeletal organisation (FRMD6, LIMK1, KIF23 and CNN1) and calcium ion binding (PVALB, CIB2, PCDH17, VCAN and CDH11) were transcriptionally more active. The present study allows for better understanding of the physiological processes associated with lipid metabolism and muscle fiber organization. This information could be helpful in further research aiming to estimate the genetic markers.

Evaluation of changes arising in the pig mesenchymal stromal cells transcriptome following cryopreservation and Trichostatin A treatment.

Cryopreservation is an important procedure in maintenance and clinical applications of mesenchymal stem/stromal cells (MSCs). Although the methods of cell freezing using various cryoprotectants are well developed and allow preserving structurally intact living cells, the freezing process can be considered as a severe cellular stress associated with ice formation, osmotic damage, cryoprotectants migration/cytotoxicity or rapid cell shrinkage. The cellular response to freezing stress is aimed at the restoring of homeostasis and repair of cell damage and is crucial for cell viability. In this study we evaluated the changes arising in the pig mesenchymal stromal cell transcriptome following cryopreservation and showed the vast alterations in cell transcriptional activity (5,575 genes with altered expression) suggesting the engagement in post-thawing cell recovery of processes connected with cell membrane tension regulation, membrane damage repair, cell shape maintenance, mitochondria-connected energy homeostasis and apoptosis mediation. We also evaluated the effect of known gene expression stimulator-Trichostain A (TSA) on the frozen/thawed cells transcriptome and showed that TSA is able to counteract to a certain extent transcriptome alterations, however, its specificity and advantages for cell recovery after cryopreservation require further studies.

Whole-genome DNA methylation characteristics in pediatric precursor B cell acute lymphoblastic leukemia (BCP ALL).

In addition to genetic alterations, epigenetic abnormalities have been shown to underlie the pathogenesis of acute lymphoblastic leukemia (ALL)-the most common pediatric cancer. The purpose of this study was to characterize the whole genome DNA methylation profile in children with precursor B-cell ALL (BCP ALL) and to compare this profile with methylation observed in normal bone marrow samples. Additional efforts were made to correlate the observed methylation patterns with selected clinical features. We assessed DNA methylation from bone marrow samples obtained from 38 children with BCP ALL at the time of diagnosis along with 4 samples of normal bone marrow cells as controls using Infinium MethylationEPIC BeadChip Array. Patients were diagnosed and stratified into prognosis groups according to the BFM ALL IC 2009 protocol. The analysis of differentially methylated sites across the genome as well as promoter methylation profiles allowed clear separation of the leukemic and control samples into two clusters. 86.6% of the promoter-associated differentially methylated sites were hypermethylated in BCP ALL. Seven sites were found to correlate with the BFM ALL IC 2009 high risk group. Amongst these, one was located within the gene body of the MBP gene and another was within the promoter region- PSMF1 gene. Differentially methylated sites that were significantly related with subsets of patients with ETV6-RUNX1 fusion and hyperdiploidy. The analyzed translocations and change of genes' sequence context does not affect methylation and methylation seems not to be a mechanism for the regulation of expression of the resulting fusion genes.

Genomic landscape of copy number variation and copy neutral loss of heterozygosity events in equine sarcoids reveals increased instability of the sarcoid genome.

Although they are the most common neoplasms in equids, sarcoids are not fully characterized at the molecular level. Therefore, the objective of this study was to characterize the landscape of structural rearrangements, such as copy number variation (CNV) and copy neutral loss of heterozygosity (cnLOH), in the genomes of sarcoid tumor cells. This information will not only broaden our understanding of the characteristics of this genome but will also improve the general knowledge of this tumor and the mechanisms involved in its generation. To this end, Equine SNP64K Illumina microarrays were applied along with bioinformatics tools dedicated for signal intensity analysis. The analysis revealed increased instability of the genome of sarcoid cells compared with unaltered skin tissue samples, which was manifested by the prevalence of CNV and cnLOH events. Many of the identified CNVs overlapped with the other research results, but the simultaneously observed variability in the number and sizes of detected aberrations indicated a need for further studies and the development of more reliable bioinformatics algorithms. The functional analysis of genes co-localized with the identified aberrations revealed that these genes are engaged in vital cellular processes. In addition, a number of these genes directly contribute to neoplastic transformation. Furthermore, large numbers of cnLOH events identified in the sarcoids suggested that they may play no less significant roles than CNVs in the carcinogenesis of this tumor. Thus, our results indicate the importance of cnLOH and CNV in equine sarcoid oncogenesis and present a direction of future research.

The effect of histone deacetylase inhibitor trichostatin A on porcine mesenchymal stem cell transcriptome.

The use of histone deacetylase inhibitors such as trichostatin A (TSA) for epigenetic transformation of mesenchymal stem cells (MSCs), whose nuclei will be transferred into enucleated oocytes, is a novel approach in research involving somatic cell cloning of pigs and other mammalian species. Although the effectiveness of TSA in cloning applications was confirmed, processes and mechanisms underlying achieved effects are not yet fully understood, especially for pig MSCs. To contribute to this knowledge, in this study we performed a comprehensive transcriptome analysis using high-throughput sequencing of pig bone-marrow derived MSCs, both treated and untreated with TSA, and evaluated the effect of TSA administration on their transcription profile after 24 h of in vitro culture. The expression of selected positive and negative mesenchymal surface antigens was also evaluated in these cells by flow cytometry. Subsequently, the stability of induced expression changes was evaluated after another 55-72 h of culture without TSA. The results of this study showed that TSA does not affect the expression of the selected surface antigens related to MSC mesenchymal stemness origin, namely: CD90 (positive marker), CD31 and CD34 (negative markers) and has a wide stimulating effect on MSCs transcription, affecting genes across the whole genome with some minor signs of site-specific acting in regions on SSC2 and SSC6. TSA turned out to have a higher impact on already expressed genes with only minor abilities to induce expression of silenced genes. Genes with expression affected by TSA were related to a wide range of biological processes, however, we found some evidence for specific stimulation of genes associated with development, differentiation, neurogenesis or myogenesis. TSA also seemed to interfere with Wnt signaling pathways by upregulation of several engaged genes. The analysis of cell transcriptome after prolonged culture following the TSA removal, showed that the expression level of majority of genes affected by TSA is restored to the initial level. Nonetheless, the set of about six hundred genes responsible for e.g. adhesion, signal transduction and cell communication was altered even after 55-72 h of culture without TSA. TSA also enhanced expression of some of pluripotency marker genes (FGF2, LIF, TERT) but their expression was stabilized during further culture without TSA. The detailed analysis of factors connected with neuron-like differentiation allowed us to assume that TSA mostly stimulates neurogenic differentiation pathway in the pig MSCs possibly through interaction with Wnt-mediated signaling and thus triggers mechanisms conducive to epigenetic reprograming.

Comprehensive characteristics of microRNA expression profile of equine sarcoids.

Equine sarcoids are the most common neoplasms occurring in horses. Despite frequent occurrence, they are still not well described at the molecular level. Thus, in the present study, we performed a comprehensive comparative analysis of sarcoid miRNAome profile to identify aberrantly expressed microRNAs, along with their structural variants, potentially useful as biomarkers and, in a wider perspective, broaden the knowledge about this tumor and underlying mechanisms. To this end, we conducted next generation sequencing and as a result we identified both known and potentially novel miRNAs. Differential expression analysis revealed the existence of almost one hundred miRNAs being over- or underexpressed in sarcoids in comparison to healthy tissue (p-adj<0.05), of which many are known for their involvement in processes crucial for neoplastic transformation. Among upregulated miRNAs there were those associated with decreased cell adhesion abilities as well as engaged in global protein production, while downregulation of some miRs i.a. increased cell expansion abilities. Moreover, we identified altered expression levels of miRNA variants (isomiRs) between the investigated tissues. Further analysis revealed that 5' isomiRs comprise different seed sequences leading to target gene switching followed by activation of different biological pathways. Our results are the first which revealed the complexity of microRNA profiles in equine sarcoids and skin tissue, along with the dynamism of their growing in importance concomitants, namely isomiRs. They also showed miRNA molecules and biological pathways important from the sarcoid oncogenesis point of view.

General assessment of copy number variation in normal and tumor tissues of the domestic dog (Canis lupus familiaris).

In recent years, characterization of a copy number variation (CNV) of the genomic DNA has provided evidence for the relationship of this type of genetic variation with the occurrence of a broad spectrum of diseases, including cancer lesions. Copy number variants (CNVs) also occur in the genomes of healthy individuals as a result of abnormal recombination processes in germ cells and have a hereditary character contributing to the natural genetic diversity. Recent image analysis methods and advanced computational techniques allow for identification of CNVs using SNPs genotyping microarrays based on the analysis of signal intensity observed for markers located in the specific genomic regions. In this study we used CanineHD BeadChip assay (Illumina) to identify both natural and cancer-induced CNVs in the genomes of different dog breeds and in different cancer types occurring in this species. The obtained results showed that structural aberrations are a common phenomenon arising during a tumor progression and are more complex and widespread in tumors of mesenchymal tissue origin than in epithelial tissue originating tumors. The tumor derived CNVs, in comparison to healthy samples, were characterized by larger sizes of regions, higher number of amplifications, and in some cases encompassed genes with potential effect on tumor progression.

Changes in DNA methylation patterns and repetitive sequences in blood lymphocytes of aged horses.

It is known that aged organisms have modified epigenomes. Epigenetic modifications, such as changes in global and locus-specific DNA methylation, and histone modifications are suspected to play an important role in cancer development and aging. In the present study, with the well-established horse aging model, we showed the global loss of DNA methylation in blood lymphocytes during juvenile-to-aged period. Additionally, we tested a pattern of DNA methylation of ribosomal DNA and selected genes such as IGF2 and found no significant changes during development and aging. We asked if genetic components such as polymorphisms within DNA methyltransferase genes, DNMT1, DNMT3a, and DNMT3b, may contribute to observed changes in global DNA methylation status. The analysis of seven intragenic polymorphisms did not reveal any significant association with changes in global DNA methylation. Telomere shortage and a loss of pericentromeric heterochromatin during juvenile-to-aged period were also observed. Transcriptional rDNA activity, assessed as the number and size of nucleolar organizer regions, reflecting physiological state of the cell, and mitotic index were decreased with increasing horse donor age. Moreover, changes during juvenile-to-aged period and adult-to-aged period were compared and discussed. Taken together, changes in global DNA methylation status originating in development and affecting the stability of repetitive sequences may be associated with previously reported genomic instability during horse aging.