Peter Dovč

Epigenetic regulation of microRNAs in cancer: An integrated review of literature

MicroRNAs (miRNAs) belong to the heterogeneous class of non-coding RNAs (ncRNAs) that regulate the translation and degradation of target mRNAs, and control approximately 30% of human genes. MiRNA genes might be silenced in human tumors (oncomiRs) by aberrant hypermethylation of CpG islands that encompass or lie adjacent to miRNA genes and/or by histone modifications. We performed literature search for research articles describing epigenetically regulated miRNAs in cancer and identified 45 studies that were published between 2006 and 7/2010. The data from those papers are fragmented and methodologically heterogeneous and our work represents first systematic review towards to integration of diverse sets of information. We reviewed the methods used for detection of miRNA epigenetic regulation, which comprise bisulfite genomic sequencing PCR (BSP), bisulfite pyrosequencing, methylation specific PCR (MSP), combined bisulfite restriction analysis (COBRA), methylation sensitive single nucleotide primer extension (Ms-SNuPE), MassARRAY technique and some modifications of those methods. This integrative study revealed 122 miRNAs that were reported to be epigenetically regulated in 23 cancer types. Compared to protein coding genes, human oncomiRs showed an order of magnitude higher methylation frequency (11.6%; 122/1048 known miRNAs). Nearly half, (45%; 55/122) epigenetically regulated miRNAs were associated with different cancer types, but other 55% (67/122) miRNAs were present in only one cancer type and therefore representing cancer-specific biomarker potential. The data integration revealed miRNA epigenomic hot spots on the chromosomes 1q, 7q, 11q, 14q and 19q. CpG island analysis of corresponding miRNA precursors (pre-miRNAs) revealed that 20% (26/133) of epigenetically regulated miRNAs had a CpG island within the range of 5kb upstream, among them 14% (19/133) of miRNAs resided within the CpG island. Our integrative survey and analyses revealed candidate cancer-specific miRNA epigenetic signatures which provide the basis for new therapeutic strategies in cancer by targeting the epigenetic regulation of miRNAs.

Database of cattle candidate genes and genetic markers for milk production and mastitis.

A cattle database of candidate genes and genetic markers for milk production and mastitis has been developed to provide an integrated research tool incorporating different types of information supporting a genomic approach to study lactation, udder development and health. The database contains 943 genes and genetic markers involved in mammary gland development and function, representing candidates for further functional studies. The candidate loci were drawn on a genetic map to reveal positional overlaps. For identification of candidate loci, data from seven different research approaches were exploited: (i) gene knockouts or transgenes in mice that result in specific phenotypes associated with mammary gland (143 loci); (ii) cattle QTL for milk production (344) and mastitis related traits (71); (iii) loci with sequence variations that show specific allele-phenotype interactions associated with milk production (24) or mastitis (10) in cattle; (iv) genes with expression profiles associated with milk production (207) or mastitis (107) in cattle or mouse; (v) cattle milk protein genes that exist in different genetic variants (9); (vi) miRNAs expressed in bovine mammary gland (32) and (vii) epigenetically regulated cattle genes associated with mammary gland function (1). Fourty-four genes found by multiple independent analyses were suggested as the most promising candidates and were further in silico analysed for expression levels in lactating mammary gland, genetic variability and top biological functions in functional networks. A miRNA target search for mammary gland expressed miRNAs identified 359 putative binding sites in 3′UTRs of candidate genes.

Neurological Symptoms in Patients Whose Cerebrospinal Fluid Is Culture- and/or Polymerase Chain Reaction-Positive for Mycoplasma pneumoniae

We describe 13 patients with neurological signs and symptoms associated with Mycoplasma pneumoniae infection. M. pneumoniae was isolated from the cerebrospinal fluid (CSF) of 9 patients: 5 with meningoencephalitis, 2 with meningitis, and 1 with cerebrovascular infarction. One patient had headache and difficulties with concentration and thinking for 1 month after the acute infection. M. pneumoniae was detected, by means of PCR, in the CSF of 4 patients with negative culture results. Two had epileptic seizures, 1 had blurred vision as a consequence of edema of the optic disk, and 1 had peripheral nerve neuropathy.

Catalog of MicroRNA Seed Polymorphisms in Vertebrates

MicroRNAs (miRNAs) are a class of non-coding RNA that plays an important role in posttranscriptional regulation of mRNA. Evidence has shown that miRNA gene variability might interfere with its function resulting in phenotypic variation and disease susceptibility. A major role in miRNA target recognition is ascribed to complementarity with the miRNA seed region that can be affected by polymorphisms. In the present study, we developed an online tool for the detection of miRNA polymorphisms (miRNA SNiPer) in vertebrates (http://www.integratomics-time.com/miRNA-SNiPer) and generated a catalog of miRNA seed region polymorphisms (miR-seed-SNPs) consisting of 149 SNPs in six species. Although a majority of detected polymorphisms were due to point mutations, two consecutive nucleotide substitutions (double nucleotide polymorphisms, DNPs) were also identified in nine miRNAs. We determined that miR-SNPs are frequently located within the quantitative trait loci (QTL), chromosome fragile sites, and cancer susceptibility loci, indicating their potential role in the genetic control of various complex traits. To test this further, we performed an association analysis between the mmu-miR-717 seed SNP rs30372501, which is polymorphic in a large number of standard inbred strains, and all phenotypic traits in these strains deposited in the Mouse Phenome Database. Analysis showed a significant association between the mmu-miR-717 seed SNP and a diverse array of traits including behavior, blood-clinical chemistry, body weight size and growth, and immune system suggesting that seed SNPs can indeed have major pleiotropic effects. The bioinformatics analyses, data and tools developed in the present study can serve researchers as a starting point in testing more targeted hypotheses and designing experiments using optimal species or strains for further mechanistic studies.

Variable expression of epitopes on the surface of Mycoplastna gattisepticum demonstrated with monoclonal antibodies

Twelve monoclonal antibodies (Mabs) against Mycoplasma gallisepticum (Mg) strains F, R, S6(208) and PET2 were used for analysis of epitopes of 22 Mg strains. Six Mabs recognized surface epitopes in the majority of strains, but did not react with variant strains like K 503 and K 703. Two Mabs reacted with epitopes on about 56 kilodalton (kDa) proteins and showing consistent expression on Mg colonies. Three Mabs recognized three different variable surface epitopes associated with about 67 kDa proteins and one Mab variable epitope on about 33 and 80 kDa proteins. Two-dimensional immunoblotting showed considerable differences in the charge of proteins bearing variable surface epitopes in different Mg strains. Subcloning of four low passage Mg strains using Mabs for screening populations that derived from a single colony with defined surface epitopes showed that some colonies may switch surface epitopes associated with 67 and 80 kDa proteins. This switching was reversible and generated subpopulations of Mg expressing different combinations of surface epitopes. Phenotypic switching of epitopes probably occurs also in vivo and may be the mechanism enabling Mg to evade the host immune response.

Genome-wide and species-wide in silico screening for intragenic MicroRNAs in human, mouse and chicken.

MicroRNAs (miRNAs) are non-coding RNAs (ncRNAs) involved in regulation of gene expression. Intragenic miRNAs, especially those exhibiting a high degree of evolutionary conservation, have been shown to be coordinately regulated and/or expressed with their host genes, either with synergistic or antagonistic correlation patterns. However, the degree of cross-species conservation of miRNA/host gene co-location is not known and co-expression information is incomplete and fragmented among several studies. Using the genomic resources (miRBase and Ensembl) we performed a genome-wide in silico screening (GWISS) for miRNA/host gene pairs in three well-annotated vertebrate species: human, mouse, and chicken. Approximately half of currently annotated miRNA genes resided within host genes: 53.0% (849/1,600) in human, 48.8% (418/855) in mouse, and 42.0% (210/499) in chicken, which we present in a central publicly available Catalog of intragenic miRNAs (http://www.integratomics-time.com/miR-host/catalog). The miRNA genes resided within either protein-coding or ncRNA genes, which include long intergenic ncRNAs (lincRNAs) and small nucleolar RNAs (snoRNAs). Twenty-seven miRNA genes were found to be located within the same host genes in all three species and the data integration from literature and databases showed that most (26/27) have been found to be co-expressed. Particularly interesting are miRNA genes located within genes encoding for miRNA silencing machinery (DGCR8, DICER1, and SND1 in human and Cnot3, Gdcr8, Eif4e, Tnrc6b, and Xpo5 in mouse). We furthermore discuss a potential for phenotype misattribution of miRNA host gene polymorphism or gene modification studies due to possible collateral effects on miRNAs hosted within them. In conclusion, the catalog of intragenic miRNAs and identified 27 miRNA/host gene pairs with cross-species conserved co-location, co-expression, and potential co-regulation, provide excellent candidates for further functional annotation of intragenic miRNAs in health and disease.

History of Lipizzan horse maternal lines as revealed by mtDNA analysis

Sequencing of the mtDNA control region (385 or 695 bp) of 212 Lipizzans from eight studs revealed 37 haplotypes. Distribution of haplotypes among studs was biased, including many private haplotypes but only one haplotype was present in all the studs. According to historical data, numerous Lipizzan maternal lines originating from founder mares of different breeds have been established during the breeds history, so the broad genetic base of the Lipizzan maternal lines was expected. A comparison of Lipizzan sequences with 136 sequences of domestic- and wild-horses from GenBank showed a clustering of Lipizzan haplotypes in the majority of haplotype subgroups present in other domestic horses. We assume that haplotypes identical to haplotypes of early domesticated horses can be found in several Lipizzan maternal lines as well as in other breeds. Therefore, domestic horses could arise either from a single large population or from several populations provided there were strong migrations during the early phase after domestication. A comparison of Lipizzan haplotypes with 56 maternal lines (according to the pedigrees) showed a disagreement of biological parentage with pedigree data for at least 11% of the Lipizzans. A distribution of haplotype-frequencies was unequal (0.2%–26%), mainly due to pedigree errors and haplotype sharing among founder mares.

Gly482Ser polymorphism of the peroxisome proliferator–activated receptor‐γ coactivator‐1 gene might be a risk factor for diabetic retinopathy in Slovene population (Caucasians) with type 2 diabetes and the Pro12Ala polymorphism of the PPARγ gene is not

The peroxisome proliferator–activated receptor‐gamma (PPARγ) gene has been recently associated with type 2 diabetes, obesity and traits depending on VEGF expression (e.g. retinopathy). The PPARγ gene and its coactivator, the peroxisome proliferator–activated receptor‐gamma coactivator‐1 (PPARGC1) gene, have been implicated to be involved in glucose uptake and altered lipid oxidation. We therefore hypothesized that the Gly482Ser polymorphism of the PPARGC1 gene and Pro12Ala polymorphism of the PPARγ gene might confer susceptibility to diabetic retinopathy in type 2 diabetes. The aim of this study was to investigate the association between the Pro12Ala polymorphism in the PPARγ gene and Gly482Ser polymorphism in the PPARGC1 gene and the development of diabetic retinopathy in the Slovene population (Caucasians) with type 2 diabetes.

Identification of chromosomal regions associated with growth and carcass traits in an F3 full sib intercross line originating from a cross of chicken lines divergently selected on body weight.

An F(3) resource population originating from a cross between two divergently selected lines for high (D+ line) or low (D- line) body weight at 8-weeks of age (BW55) was generated and used for Quantitative Trait Locus (QTL) mapping. From an initial cross of two founder F(0) animals from D(+) and D(-) lines, progeny were randomly intercrossed over two generations following a full sib intercross line (FSIL) design. One hundred and seventy-five genome-wide polymorphic markers were employed in the DNA pooling and selective genotyping of F(3) to identify markers with significant effects on BW55. Fifty-three markers on GGA2, 5 and 11 were then genotyped in the whole F(3) population of 503 birds, where interval mapping with GridQTL software was employed. Eighteen QTL for body weight, carcass traits and some internal organ weights were identified. On GGA2, a comparison between 2-QTL vs. 1-QTL analysis revealed two separate QTL regions for body, feet, breast muscle and carcass weight. Given co-localization of QTL for some highly correlated traits, we concluded that there were 11 distinct QTL mapped. Four QTL localized to already mapped QTL from other studies, but seven QTL have not been previously reported and are hence novel and unique to our selection line. This study provides a low resolution QTL map for various traits and establishes a genetic resource for future fine-mapping and positional cloning in the advanced FSIL generations.

Genome-wide in silico screening for microRNA genetic variability in livestock species

MicroRNAs are a class of non-coding RNAs that post-transcriptionally regulate target gene expression. Previous studies have shown that microRNA gene variability can interfere with its function, resulting in phenotypic variation. Polymorphisms within microRNA genes present a source of novel biomarkers for phenotypic traits in animal breeding. However, little is known about microRNA genetic variability in livestock species, which is also due to incomplete data in genomic resource databases. Therefore, the aim of this study was to perform a genome-wide in silico screening of genomic sources and determine the genetic variability of microRNA genes in livestock species using mirna sniper 3.0 (http://www.integratomics-time.com/miRNA-SNiPer/), a new version of our previously developed tool. By examining Ensembl and miRBase genome builds, it was possible to design a tool-based generated search of 16 genomes including four livestock species: pig, horse, cattle and chicken. The analysis revealed 65 polymorphisms located within mature microRNA regions in these four species, including 28% within the seed region in cattle and chicken. Polymorphic microRNA genes in cattle and chicken were further examined for mapping to quantitative trait loci regions associated with production and health traits. The developed bioinformatics tool enables the analysis of polymorphic microRNA genes and prioritization of potential regulatory polymorphisms and therefore contributes to the development of microRNA-based biomarkers in livestock species. The assembled catalog and the developed tool can serve the animal science community to efficiently select microRNA SNPs for further quantitative and molecular genetic evaluations of their phenotypic effects and causal associations with livestock production traits.