Twelve toll-like receptor (TLR) genes in the family Equidae - comparative genomics, selection and evolution.

Toll-like receptors (TLRs) represent an important part of the innate immune system. While human and murine TLRs have been intensively studied, little is known about TLRs in non-model species. The order Perissodactyla comprises a variety of free-living and domesticated species exposed to different pathogens in different habitats and is therefore suitable for analyzing the diversity and evolution of immunity-related genes. We analyzed TLR genes in the order Perissodactyla with a focus on the family Equidae. Twelve TLRs were identified by bioinformatic analyses of online genomic resources; their sequences were confirmed in equids by genomic DNA re-sequencing of a panel of nine species. The expression of TLR11 and TLR12 was confirmed in the domestic horse by cDNA sequencing. Phylogenetic reconstruction of the TLR gene family in Perissodactyla identified six sub-families. TLR4 clustered together with TLR5; the TLR1-6-10 subfamily showed a high degree of sequence identity. The average estimated evolutionary divergence of all twelve TLRs studied was 0.3% among the Equidae; the most divergent CDS were those of Equus caballus and Equus hemionus kulan (1.34%) in the TLR3, and Equus africanus somaliensis and Equus quagga antiquorum (2.1%) in the TLR1 protein. In each TLR gene, there were haplotypes shared between equid species, most extensively in TLR3 and TLR9 CDS, and TLR6 amino acid sequence. All twelve TLR genes were under strong negative overall selection. Signatures of diversifying selection in specific codon sites were detected in all TLRs except TLR8. Differences in the selection patterns between virus-sensing and non-viral TLRs were observed.

Associations between the presence of specific antibodies to the West Nile Virus infection and candidate genes in Romanian horses from the Danube delta.

The West Nile virus (WNV) is a mosquito-borne flavivirus causing meningoencephalitis in humans and animals. Due to their particular susceptibility to WNV infection, horses serve as a sentinel species. In a population of Romanian semi-feral horses living in the Danube delta region, we have analyzed the distribution of candidate polymorphic genetic markers between anti WNV-IgG seropositive and seronegative horses. Thirty-six SNPs located in 28 immunity-related genes and 26 microsatellites located in the MHC and LY49 complex genomic regions were genotyped in 57 seropositive and 32 seronegative horses. The most significant association (pcorr < 0.0002) was found for genotypes composed of markers of the SLC11A1 and TLR4 genes. Markers of five other candidate genes (ADAM17, CXCR3, IL12A, MAVS, TNFA), along with 5 MHC class I and LY49-linked microsatellites were also associated with the WNV antibody status in this model horse population. The OAS1 gene, previously associated with WNV-induced clinical disease, was not associated with the presence of anti-WNV antibodies.

Genetic susceptibility to West Nile virus infection in Camargue horses.

West Nile virus (WNV) is a mosquito-borne zoonotic neurotropic virus capable to cause lethal meningoencephalitis (WNE) in infected hosts such as birds, horses, and humans. Due to their sensitivity, horses serve as sentinel species in areas at risk. We studied a population of Camargue horses living in Southern France in two zones with endemic WNV circulation where WNV outbreaks were recorded in 2000 and 2003-4. Two sets of microsatellite markers located in MHC and Ly49 genomic regions were genotyped as well as multiple SNPs in ten immunity-related candidate gene regions. Associations between genetic polymorphisms and resistance/susceptibility to WNE were tested. While single marker associations were weak, compound two-gene genotypes of SNPs located within the MAVS, NCR2 and IL-10 genes and microsatellites HMS082 and CZM013 were associated with susceptibility to WNE. Combinations of microsatellite markers CZM009, ABGe17402 and ABGe9019 were associated with simple seroconversion without clinical signs of WNE (resistance). In addition, a distribution of polymorphic markers between WNV-IgG seropositive horses and a control group of WNV-IgG seronegative horses was tested. One SNP in the OAS1 gene (NC_009151.3:g.21961328A>G) was significantly associated with the seropositive phenotype (pcorr = 0.023; OR = 40.5 CI (4.28; 383.26); RR = 8.18 CI (1.27; 52.89) in the Camargue breed. In compound genotypes, SNP markers for SLC11A1, MAVS, OAS1, TLR4, ADAM17 and NCR2 genes and ten microsatellites showed non-random distribution between seropositive and seronegative groups of horses. Further analysis of associated markers could contribute to our understanding of anti-WNV defense mechanisms in horses.

Microsatellite markers for evaluating the diversity of the natural killer complex and major histocompatibility complex genomic regions in domestic horses.

Genotyping microsatellite markers represents a standard, relatively easy, and inexpensive method of assessing genetic diversity of complex genomic regions in various animal species, such as the major histocompatibility complex (MHC) and/or natural killer cell receptor (NKR) genes. MHC-linked microsatellite markers have been identified and some of them were used for characterizing MHC polymorphism in various species, including horses. However, most of those were MHC class II markers, while MHC class I and III sub-regions were less well covered. No tools for studying genetic diversity of NKR complex genomic regions are available in horses. Therefore, the aims of this work were to establish a panel of markers suitable for analyzing genetic diversity of the natural killer complex (NKC), and to develop additional microsatellite markers of the MHC class I and class III genomic sub-regions in horses. Nine polymorphic microsatellite loci were newly identified in the equine NKC. Along with two previously reported microsatellites flanking this region, they constituted a panel of 11 loci allowing to characterize genetic variation in this functionally important part of the horse genome. Four newly described MHC class I/III-linked markers were added to 11 known microsatellites to establish a panel of 15 MHC markers with a better coverage of the class I and class III sub-regions. Major characteristics of the two panels produced on a group of 65 horses of 13 breeds and on five Przewalski's horses showed that they do reflect genetic variation within the horse species.

Candidate gene molecular markers as tools for analyzing genetic susceptibility to morbillivirus infection in stranded Cetaceans.

Morbilliviruses, such as Cetacean morbillivirus (CeMV) or Phocine distemper virus (PDV), represent a growing threat for marine mammals on both hemispheres. Because free-ranging animal populations strongly rely on natural resistance mechanisms, innate immunity-related genes and virus cell entry receptor genes may represent key factors involved in susceptibility to CeMV in Cetaceans. Using the next generation sequencing technology, we have sequenced 11 candidate genes in two model species, Stenella coeruleoalba and Phocoena phocoena. Suitable single nucleotide polymorphism markers of potential functional importance, located in genes coding for basigin (BSG, CD147), the signaling lymphocyte activating molecule (SLAMF1), the poliovirus-related receptor-4 (NECTIN4, PVRL4), toll-like receptors 3, 7, 8 (TLR3, TLR7, TLR8), natural resistance-associated macrophage protein (SLC11A1) and natural cytotoxicity triggering receptor 1 (NCR1), were identified in each model species, along with MHC-DQB haplotypes unique for each species. This set of molecular markers represents a potentially useful tool for studying host genetic variation and susceptibility to morbillivirus infection in Cetaceans as well as for studying functionally important genetic diversity of selected Cetacean populations.

Worldwide frequency distribution of the 'Gait keeper' mutation in the DMRT3 gene.

For centuries, domestic horses have represented an important means of transport and served as working and companion animals. Although their role in transportation is less important today, many horse breeds are still subject to intense selection based on their pattern of locomotion. A striking example of such a selected trait is the ability of a horse to perform additional gaits other than the common walk, trot and gallop. Those could be four-beat ambling gaits, which are particularly smooth and comfortable for the rider, or pace, used mainly in racing. Gaited horse breeds occur around the globe, suggesting that gaitedness is an old trait, selected for in many breeds. A recent study discovered that a nonsense mutation in DMRT3 has a major impact on gaitedness in horses and is present at a high frequency in gaited breeds and in horses bred for harness racing. Here, we report a study of the worldwide distribution of this mutation. We genotyped 4396 horses representing 141 horse breeds for the DMRT3 stop mutation. More than half (2749) of these horses also were genotyped for a SNP situated 32 kb upstream of the DMRT3 nonsense mutation because these two SNPs are in very strong linkage disequilibrium. We show that the DMRT3 mutation is present in 68 of the 141 genotyped horse breeds at a frequency ranging from 1% to 100%. We also show that the mutation is not limited to a geographical area, but is found worldwide. The breeds with a high frequency of the stop mutation (>50%) are either classified as gaited or bred for harness racing.

Genetics of anti-EHV antibody responses in a horse population.

Individual variation in immune responses to herpesviruses was observed in various species. Here, associations between polymorphic molecular markers and life-long anti-EHV-1/4 antibody immune responses were analyzed in a model EHV-infected population of the Old Kladruber horses. Two-dimensional analysis including overall mean titers and titer dynamics expressed by differences between spring and autumn titers allowed identification of low-responders. 50 randomly selected microsatellites and nine single nucleotide polymorphisms in nine immunity-related candidate genes were genotyped. Due to differences (p<0.001) in antibody titers between two color varieties of Old Kladruber horses, separate association studies were performed in the two sub-populations by using the Fisher's exact test. In black horses, the interleukin 4 receptor and MxA protein coding genes, and the microsatellite TKY325 were associated with the responder status. In the grey population, the microsatellite TKY343 showed significant association with anti-EHV antibody responsiveness after Bonferroni corrections.

Effect of genetic variation in the MHC Class II DRB region on resistance and susceptibility to Johne's disease in endangered Indian Jamunapari goats.

The pathogenesis of Johne's disease (JD), caused by Mycobacterium avium subsp. paratuberculosis (MAP), is complex and has not been completely understood yet. In the present study, we analysed the polymorphism in the exon-2 of the caprine major histocompatibility complex (MHC) Class II DRB region and its association with resistance or susceptibility to JD. A total of 203 Jamunapari goats, which is an Indian endangered breed highly susceptible to JD, kept at a single farm were studied. On the basis of clinical signs, microscopic examination, faecal culture, ELISA and diagnostic PCR, 60 and 143 goats were classified as resistant and susceptible to JD, respectively. PCR-based restriction fragment length polymorphism (PCR-RFLP) with two enzymes, PstI and TaqI, was used to assess variation in the DRB gene(s) in all 203 goats studied. Two di-allelic single nucleotide polymorphisms (SNPs), here referred as 'P' and 'T', were tested. In each of them, three genotypes were found in the group analysed. The minimum allele frequencies (MAFs) were 0.233 and 0.486 for the P and T SNPs, respectively. Statistically significant associations between alleles, individual genotypes and composed genotypes of both SNPs were found. The frequency of p and t alleles, of individual pp and tt and of composed pptt genotypes were significantly higher (P(corr) < 0.001) in the 'resistant' group as compared to the 'susceptible' group, while the P and T alleles were associated with susceptibility (P(corr) < 0.001). In heterozygous genotypes, susceptibility was dominant over resistance. The effects of both SNP on resistance and susceptibility were comparable and composed heterozygous genotypes showed intermediate levels of susceptibility in terms of the odds ratio and P-values calculated.

Immunity-related gene single nucleotide polymorphisms associated with Rhodococcus equi infection in foals.

In previous work, we found significant associations of horse polymorphic microsatellite and immunity-related (IR) gene markers with Rhodococcus equi infection of foals. Here, a statistically significant association between a single nucleotide polymorphism (SNP) within the interleukin 7 receptor-encoding gene (IL7R) with high R. equi burden in transtracheal aspirates was found (Fisher's F = 0.043, odds ratio: 8.00, 95% confidence interval: 1.127-56.795). Further positional and/or functional candidate genes investigated TLR2, IL13, IL17A, IL28R, TACE/ADAM 17 and GBP1, were not associated with infection in this study. SNPs analysed were found by sequencing and appropriate restriction fragment length polymorphism markers were developed. Their associations with R. equi infection were tested by genotyping thoroughbred foals from the original study. The association was confirmed by analysing genotypes composed with genes previously reported to be associated with R. equi infection in the same group.

Single nucleotide polymorphisms of interleukin-1 beta related genes and their associations with infection in the horse.

In previous work, we found significant associations of horse chromosome 15 (ECA15) microsatellite markers HMSO1 and HTG06 with two horse infections, Rhodococcus equi and Lawsonia intracellularis, respectively. Interleukin-1 beta subunit and interleukin-1 receptor antagonist encoding genes (IL1B and IL1RN) could be considered as candidate genes underlying the associations reported. Therefore, we identified single nucleotide polymorphisms (SNPs) within three interleukin-1 beta functionally related genes: IL1B, IL1RN and Casp1 (interleukin-1 beta converting enzyme/caspasel encoding gene). Using appropriate restriction fragment length polymorphism (PCR-RFLP) and/or single strand conformation polymorphism (PCR-SSCP) markers, their associations with the two infections by genotyping foals from the original study were tested. In addition, the physical localization of one of the two closely located genes, IL1RN, was re-assessed by fluorescence in-situ hybridization (FISH). A statistically significant association between an intronic SNP of the Casp1 gene with R. equi infection was found. The IL1RN gene was localized to 15q13-q14 in agreement with its originally reported physical position.

Report of the 3rd Havemeyer workshop on allergic diseases of the Horse, Hólar, Iceland, June 2007.

Allergic diseases occur in most mammals, although some species such as humans, dogs and horses seem to be more prone to develop allergies than others. In horses, insect bite hypersensitivity (IBH), an allergic dermatitis caused by bites of midges, and recurrent airway obstruction (RAO), a hyperreactivity to stable born dust and allergens, are the two most prevalent allergic diseases. Allergic diseases involve the interaction of three major factors: (i) genetic constitution, (ii) exposure to allergens, and (iii) a dysregulation of the immune response determined by (i) and (ii). However, other environmental factors such as infectious diseases, contact with endotoxin and degree of infestation with endoparasites have been shown to influence the prevalence of allergic diseases in humans. How these factors may impact upon allergic disease in the horse is unknown at this time. The 3rd workshop on Allergic Diseases of the Horse, with major sponsorship from the Havemeyer Foundation, was held in Hólar, Iceland, in June 2007 and focussed on immunological and genetic aspects of IBH and RAO. This particular venue was chosen because of the prevalence of IBH in exported Icelandic horses. The incidence of IBH is significantly different between Icelandic horses born in Europe or North America and those born in Iceland and exported as adults. Although the genetic factors and allergens are the same, exported adult horses show a greater incidence of IBH. This suggests that environmental or epigenetic factors may contribute to this response. This report summarizes the present state of knowledge and summarizes important issues discussed at the workshop.

Karyotypic relationships among Equus grevyi, Equus burchelli and domestic horse defined using horse chromosome arm-specific probes.

Using laser microdissection we prepared a set of horse chromosome arm-specific probes. Most of the probes were generated from horse chromosomes, some of them were derived from Equus zebra hartmannae. The set of probes were hybridized onto E. grevyi chromosomes in order to establish a genome-wide chromosomal correspondence between this zebra and horse. The use of arm-specific probes provided us with more information on the mutual arrangement of the genomes than we could obtain by means of whole-chromosome paints generated by flow sorting, even if we used reciprocal painting with probe sets from both species. By comparison of our results and results of comparative mapping in E. burchelli, we also established the chromosomal correspondence between E. grevyi and E. burchelli, providing evidence for a very close karyotypic relationship between these two zebra species. Establishment of the comparative map for E. grevyi contributes to the knowledge of the karyotypic phylogeny in the Equidae family.

Cytogenetic mapping of immunity-related genes in the domestic horse.

Chromosomal locations of 19 horse immunity-related loci (CASP1, CD14, EIF5A, FCER1A, IFNG, IL12A, IL12B, IL12RB2, IL1A, IL23A, IL4, IL6, MMP7, MS4A2, MYD88, NOS2A, PTGS2, TFRC and TLR2) were determined by fluorescence in situ hybridization. For IFNG and PTGS2, this study is a confirmation of their previously reported position. In addition, microsatellite (HMBr1) was localized in the same region as IFNG. All genes were assigned to regions of conserved synteny and the data obtained in this study enhance the comparative human-horse map. Cytogenetic localization of IL6 to ECA4q14-q21.1 suggested a new breakage point that changes the order of loci compared with HSA7. The map assignments of these loci serve as anchors for other loci and will aid in the search for candidate genes associated with traits in the horse.

Single nucleotide polymorphisms in four functionally related immune response genes in the horse: CD14,TLR4, Cepsilon, andFcepsilon R1 alpha.

The objective of this study was to identify single nucleotide polymorphisms (SNPs) within four functionally related immune response genes in the horse, and to develop genotyping techniques that could be useful for future genomic studies of horse infectious and allergic diseases. The genes analysed were: the lipopolysaccharide (LPS) receptor gene CD14, the toll-like receptor 4 gene TLR4, the gene Cepsilon encoding the IgE heavy chain molecule and the gene FcepsilonR1 alpha coding for the alpha subunit of the IgE receptor molecule. Horse-specific primers amplifying selected gene regions were designed and SNPs were searched by selective resequencing and/or by PCR-SSCP (polymerase chain reaction-sequence specific conformational polymorphism) or PCR-RFLP (PCR-restriction fragment length polymorphism). Gene expression was analysed by RT-PCR (reverse transcriptase-PCR) of all four genes examined. For CD14, the cDNA sequence was determined and a novel sequence of the 5'UTR region was identified. The protein-coding sequence was identical to that previously deposited in GenBank. 5'UTR, intronic and both synonymous and non-synonymous exonic SNPs were identified. Three SNPs were found in the CD14 gene, four in the TLR4 gene; two SNPs were identified in the Cepsilon gene, and one SNP was found in the FcepsilonR1 alpha gene. PCR-RFLP was developed for genotyping eight of the SNPs identified. The RT-PCR assay showed that all the SNPs reported here are parts of expressed genes. The results showed that important immunity-related genes in horses are polymorphic and that even non-synonymous SNPs with potential functional impact may occur. The methods developed for genotyping and haplotyping the SNPs identified represent, along with markers described previously, a potentially useful tool for genomic analysis of the function and role of these genes in immunity and in mechanisms of disease.

Characterization of the NRAMP1 (SLC11A1) gene in the horse (Equus caballus L.).

The complete coding cDNA sequence of the horse NRAMP1 (SLC11A1) gene was determined (GenBank accession number AF354445). The nucleotide sequence of the horse NRAMP1 gene is similar to sequences of this gene in other species. The gene contains 15 exons whose total length of 1,635 bp corresponds to 544 amino acids constituting the resulting putative protein. Hydrophobicity profile analysis of the deduced horse NRAMP1 gene product showed a nearly identical structure with the mouse NRAMP1 protein. The gene was found to be located on the short arm of ECA 6p12-13 by fluorescence in situ hybridization (FISH) analysis. Five allelic variants of the 5' untranslated region (UTR) were identified at the nucleotide sequence level. PCR-RFLP polymorphisms for NlaIII, TaqI, MspI and AciI were detected. Four out of five alleles could be detected using TaqI and MspI restriction enzymes. Their haplotype frequencies were different in four genetically distinct horse breeds.

Two bi-allelic single nucleotide polymorphisms within the promoter region of the horse tumour necrosis factor alpha gene.

Primers based on GenBank sequences within the 5' untranslated region (UTR) of the human and horse tumour necrosis factor alpha (TNF-alpha) genes were designed and used to amplify a 522-bp product. Sequencing of five clones derived from five independent PCRs obtained from three different animals of three different breeds (Old Kladruber, Akhal-Teke and Shetland Pony) revealed a high level of sequence identity to the TNF-alpha promoter regions of other species. The existing GenBank horse sequences were confirmed and extended upstream by 230 nucleotides. Based on the sequence obtained, a new horse-specific forward primer was designed to amplify a 213-bp PCR product, which was screened for polymorphism using single-strand conformation polymorphism (SSCP). Three allelic variants of the horse TNF-alpha gene were identified and sequenced (GenBank accession numbers ADF 349558-60). Two single nucleotide polymorphisms explained the existence of the three SSCP alleles detected: C/T and T/C single base pair substitutions at positions 137 and 147, respectively. Differences in allelic frequencies between Old Kladruber and Akhal-Teke breeds were observed.

A second locus and new alleles in the major histocompatibility complex class II (ELA-DQB) region in the horse.

More than two nucleotide sequences of the second exon of the ELA-DQB region retrieved from a single animal and two different sequences isolated from horses homozygous in the major histocompatibility complex (MHC) region by descent indicated the existence of at least two ELA-DQB loci at the genomic level. New alleles detected by polymerase chain reaction single strand conformation polymorphism (SSCP) and defined by nucleotide sequencing of the second exon of the DQB gene(s) were described. Based on the level of nucleotide sharing, at least two groups of alleles were shown to exist. The newly defined alleles belonged preferentially to one of the groups. However, their specific locus assignment was not possible from the data collected. At least one of these alleles was shown to be transcribed. No frame-shift mutations were identified among the new alleles, although one pseudoallele containing a stop codon was identified at the genomic DNA level.

[Biological principles of chemotherapy: cellular response as a result of its evolutionary memory]. / Biologický princip chemoterapie: reakce bunky jako výsledek její evolucní pameti.

The cell reaction to chemotherapy is based on certain general biological principles. The genetic structure of cell populations, their actual reaction to chemotherapy and the prognosis of their development are not accidental. They are determined by previous exposures to chemotherapeutic agents and thus their evolutional experience on Darwinian principles. Chemotherapy occurs always in a defined environment of the host with his active participation. A genetic variability in the host reaction to chemotherapeutic interference also exists and which in the long-term perspective is also of evolutional nature. The complex of evaluation of therapeutic procedures thus involves evaluation and influencing of the host and his variability (immunotherapy). Understanding of molecular mechanisms of the pathogenesis of disease, definition of specific genes, their expression, the role of their products and definition of possible targets of chemotherapy is at present a realistic goal of application of genomics and proteogenomics in medicine. The new philosophy in medicine is thus based on respecting individuality in diagnosis, treatment and prognosis. We only can wish that the trend of respecting individuality will be applied in the future, and not only in medicine.