Review: Long non-coding RNA in livestock.

Less than 2% of mammalian genomes code for proteins, but 'the majority of its bases can be found in primary transcripts' - a phenomenon termed the pervasive transcription, which was first reported in 2007. Even though most of the transcripts do not code for proteins, they play a variety of biological functions, with regulation of gene expression appearing as the most common one. Those transcripts are divided into two groups based on their length: small non-coding RNAs, which are maximally 200 bp long, and long non-coding RNAs (lncRNAs), which are longer than 200 nucleotides. The advances in next-generation sequencing methods provided a new possibility of investigating the full set of RNA molecules in the cell. In this review, we summarized the current state of knowledge on lncRNAs in three major livestock species - Sus scrofa, Bos taurus and Gallus gallus, based on the literature and the content of biological databases. In the NONCODE database, the largest number of identified lncRNA transcripts is available for pigs, but cattle have the largest number of lncRNA genes. Poultry is represented by less than a half of records. Genomic annotation of lncRNAs showed that the majority of them are assigned to introns (pig, poultry) or intergenic (cattle). The comparison with well-annotated human and mouse genomes indicates that such annotation is a result of lack of proper lncRNA annotation data. Since lncRNAs play an important role in genomic studies, their characterization in farm animals' genomes is critical in bridging the gap between genotype and phenotype.

The genetic background of clinical mastitis in Holstein-Friesian cattle.

Mastitis is an inflammatory disease of the mammary gland, which has a significant economic impact and is an animal welfare concern. This work examined the association between single nucleotide polymorphisms (SNPs) and copy number variations (CNVs) with the incidence of clinical mastitis (CM). Using information from 16 half-sib pairs of Holstein-Friesian cows (32 animals in total) we searched for genomic regions that differed between a healthy (no incidence of CM) and a mastitis-prone (multiple incidences of CM) half-sib. Three cows with average sequence depth of coverage below 10 were excluded, which left 13 half-sib pairs available for comparisons. In total, 191 CNV regions were identified, which were deleted in a mastitis-prone cow, but present in its healthy half-sib and overlapped in at least nine half-sib pairs. These regions overlapped with exons of 46 genes, among which APP (BTA1), FOXL2 (BTA1), SSFA2 (BTA2), OTUD3 (BTA2), ADORA2A (BTA17), TXNRD2 (BTA17) and NDUFS6 (BTA20) have been reported to influence CM. Moreover, two duplicated CNV regions present in nine healthy individuals and absent in their mastitis-affected half-sibs overlapped with exons of a cholinergic receptor nicotinic α 10 subunit on BTA15 and a novel gene (ENSBTAG00000008519) on BTA27. One CNV region deleted in nine mastitis-affected sibs overlapped with two neighbouring long non-coding RNA sequences located on BTA12. Single nucleotide polymorphisms with differential genotypes between a healthy and a mastitis-affected sib included 17 polymorphisms with alternate alleles in eight affected and healthy half-sib families. Three of these SNPs were located introns of genes: MET (BTA04), RNF122 (BTA27) and WRN (BTA27). In summary, structural polymorphisms in form of CNVs, putatively play a role in susceptibility to CM. Specifically, sequence deletions have a greater effect on reducing resistance against mastitis, than sequence duplications have on increasing resistance against the disease.

Differences in Cardiovascular Manifestation of Marfan Syndrome Between Children and Adults.

Marfan syndrome (MFS) is a connective tissue disorder characterized by a broad range of clinical manifestations. Cardiovascular involvement is the most life-threatening aspect of the syndrome. Although abnormalities within the cardiovascular system in adults are well documented, there is still a paucity of data regarding manifestation of MFS in childhood. The aim of the study was to compare cardiovascular manifestation of MFS between children and adults. The study population consisted of 236 patients (144 children and 92 adults), who were referred to our department with suspicion of MFS. All patients underwent complete clinical evaluation in order to confirm the diagnosis of MFS according to the modified Ghent criteria. MFS was diagnosed in 101 (44 children and 57 adults) out of the 236 patients. The other patients were diagnosed with Ehlers-Danlos syndrome, Loeys-Dietz syndrome, MASS phenotype, ectopia lentis syndrome, marfanoid habitus and other rare syndromes. The most common cardiovascular abnormality was aortic root dilatation (81.19% of patients). It was found that both adults and children had similar high rates of aortic root dilatation. Similarly, there was no significant difference with regard to the prevalence of aortic valve regurgitation and mitral valve prolapse among children and adults. These findings equivocally indicate that the aforementioned abnormalities develop in early childhood, therefore, they may be used in the early identification of patients with MFS. Other assessed abnormalities, which included mitral valve regurgitation, pulmonary artery dilation, aneurysms of aortic arch, descending thoracic aorta and abdominal aorta were found mostly in adults, and thus, are of less use in the early detection of MFS.

Landscape of copy number variations in Bos taurus: individual - and inter-breed variability.

BACKGROUND: The number of studies of Copy Number Variation in cattle has increased in recent years. This has been prompted by the increased availability of data on polymorphisms and their relationship with phenotypes. In addition, livestock species are good models for some human phenotypes. In the present study, we described the landscape of CNV driven genetic variation in a large population of 146 individuals representing 13 cattle breeds, using whole genome DNA sequence. RESULTS: A highly significant variation among all individuals and within each breed was observed in the number of duplications (P < 10-15) and in the number of deletions (P < 10-15). We also observed significant differences between breeds for duplication (P = 0.01932) and deletion (P = 0.01006) counts. The same variation CNV length - inter-individual and inter-breed differences were significant for duplications (P < 10-15) and deletions (P < 10-15). Moreover, breed-specific variants were identified, with the largest proportion of breed-specific duplications (9.57%) found for Fleckvieh and breed-specific deletions found for Brown Swiss (5.00%). Such breed-specific CNVs were predominantly located in intragenic regions, however in Simmental, one deletion present in five individuals was found in the coding sequence of a novel gene ENSBTAG00000000688 on chromosome 18. In Brown Swiss, Norwegian Red and Simmental breed-specific deletions were located within KIT and MC1R genes, which are responsible for a coat colour. The functional annotation of coding regions underlying the breed-specific CNVs showed that in Norwegian Red, Guernsey, and Simmental significantly under- and overrepresented GO terms were related to chemical stimulus involved in sensory perception of smell and the KEGG pathways for olfactory transduction. In addition, specifically for the Norwegian Red breed, the dopaminergic synapse KEGG pathway was significantly enriched within deleted parts of the genome. CONCLUSIONS: The CNV landscape in Bos taurus genome revealed by this study was highly complex, with inter-breed differences, but also a significant variation within breeds. The former, may explain some of the phenotypic differences among analysed breeds, and the latter contributes to within-breed variation available for selection.

Analysis of copy number variations in Holstein-Friesian cow genomes based on whole-genome sequence data.

Thirty-two whole genome DNA sequences of cows were analyzed to evaluate inter-individual variability in the distribution and length of copy number variations (CNV) and to functionally annotate CNV breakpoints. The total number of deletions per individual varied between 9,731 and 15,051, whereas the number of duplications was between 1,694 and 5,187. Most of the deletions (81%) and duplications (86%) were unique to a single cow. No relation between the pattern of variant sharing and a family relationship or disease status was found. The animal-averaged length of deletions was from 5,234 to 9,145 bp and the average length of duplications was between 7,254 and 8,843 bp. Highly significant inter-individual variation in length and number of CNV was detected for both deletions and duplications. The majority of deletion and duplication breakpoints were located in intergenic regions and introns, whereas fewer were identified in noncoding transcripts and splice regions. Only 1.35 and 0.79% of the deletion and duplication breakpoints were observed within coding regions. A gene with the highest number of deletion breakpoints codes for protein kinase cGMP-dependent type I, whereas the T-cell receptor α constant gene had the most duplication breakpoints. The functional annotation of genes with the largest incidence of deletion/duplication breakpoints identified 87/112 Kyoto Encyclopedia of Genes and Genomes pathways, but none of the pathways were significantly enriched or depleted with breakpoints. The analysis of Gene Ontology (GO) terms revealed that a cluster with the highest enrichment score among genes with many deletion breakpoints was represented by GO terms related to ion transport, whereas the GO term cluster mostly enriched among the genes with many duplication breakpoints was related to binding of macromolecules. Furthermore, when considering the number of deletion breakpoints per gene functional category, no significant differences were observed between the "housekeeping" and "strong selection" categories, but genes representing the "low selection pressure" group showed a significantly higher number of breakpoints.

Review of alignment and SNP calling algorithms for next-generation sequencing data.

Application of the massive parallel sequencing technology has become one of the most important issues in life sciences. Therefore, it was crucial to develop bioinformatics tools for next-generation sequencing (NGS) data processing. Currently, two of the most significant tasks include alignment to a reference genome and detection of single nucleotide polymorphisms (SNPs). In many types of genomic analyses, great numbers of reads need to be mapped to the reference genome; therefore, selection of the aligner is an essential step in NGS pipelines. Two main algorithms-suffix tries and hash tables-have been introduced for this purpose. Suffix array-based aligners are memory-efficient and work faster than hash-based aligners, but they are less accurate. In contrast, hash table algorithms tend to be slower, but more sensitive. SNP and genotype callers may also be divided into two main different approaches: heuristic and probabilistic methods. A variety of software has been subsequently developed over the past several years. In this paper, we briefly review the current development of NGS data processing algorithms and present the available software.

An optimization of hematopoietic stem and progenitor cell isolation for scientific and clinical purposes by the application of a new parameter determining the hematopoietic graft efficacy.

The transplantation of hematopoietic stem and progenitor cells (HSPC) is an established lifesaving therapy. Bone marrow (BM), harvested from heparinized cadaveric organ donors, peripheral blood (PB) and cord blood (CB), are important sources of hematopoietic stem cells. HSPCs, which are used for transplantation purposes, are routinely evaluated in terms of number of mononuclear cells (MNCs), CD34+ MNCs count and viability. The efficacy of grafting is determined additionally in clonogenic tests in vitro. These tests deliver important information about the number of HSPCs and their proliferative potential. Unfortunately, they do not give a possibility to evaluate the functional HSPC chemotactic reactivity in the SDF-1 gradient, which is probably the key phenomenon for HSPC homing after transplantation procedure. Thus, the aim of our study was to optimize HSPC isolation according to their chemotactic reactivity in SDF-1 gradient. Using multiparameter cell sorter (FACS Aria, BD) we examined the HSPCs attracted by SDF-1 on a single cell level. The population of cells which participated in the chemotactic process was highly enriched in CXCR4+lin-AC133+CD45+ cells (referred as hematopoietic stem cells) and to our surprise in CXCR4+lin-AC133+CD45- cells (referred as pluripotent stem cells) in quantitative amounts. Since reactivity of HSPCs may depend on various factors involved in the protocol of their isolation and short-term storage, we tested the most commonly used anticoagulants (ACD, CPDA-1, EDTA and Heparin) and culture media (DME, IMDM, RPMI). HSPCs, harvested from CB, PB and BM, were subsequently investigated for clonogenic growth of CFU-GM in methylcellulose cultures and for the level of apoptosis by employing annexin V staining. Evaluating clonogenic potential, ability of chemotactic reactivity in SDF-1 gradient and intensification of apoptosis of HSPC as the most safe anticoagulant and medium were selected. This study has proved that chemotactic reactivity of HSPCs is a new but very important parameter which should be included in the procedure of their isolation.

Arginase as a useful factor for the diagnosis of colorectal cancer liver metastases.

The present work is a continuation of studies on arginase as a marker in the diagnosis of colorectal cancer liver metastases (CRCLM). The purpose of the study was the evaluation of the arginase test in comparison with other colorectal cancer tests such as CEA, CA 19-9 and biochemical markers of liver function such as aspartate aminotransferase (AST) and alanine aminotransferase (ALT). The studies were conducted on blood serum from 85 patients with CRCLM obtained one to two days before tumor resection. The control group comprised 140 healthy blood donors and 81 patients with various non-malignant gastrointestinal diseases. Raised arginase activity was observed in serum of 85% of CRCLM patients, whereas elevated levels of CEA and CA 19-9 were found in 63% and 42% of patients, respectively. The combination of CEA or CA 19-9 with the arginase assay improved their sensitivity, but the sensitivity of the combined parameters was not higher than that of the arginase test itself. AST and ALT activities were increased in about 30% of CRCLM patients. The specificity of the arginase test calculated for 221 control subjects was 76%. It can thus be concluded that the determination of serum arginase activity can be helpful in the diagnosis of patients with colorectal cancer liver metastases.