Comparative karyotype analysis of the red brocket deer (M. americana sensu lato and M. rufa) complex: evidence of drastic chromosomal evolution and implications on speciation process.

Chromosomal rearrangements are often associated with playing a role in the speciation process. However, the underlying mechanism that favors the genetic isolation associated with chromosomal changes remains elusive. In this sense, the genus Mazama is recognized by its high level of karyotype diversity among species with similar morphology. A cryptic species complex has been identified within the genus, with the red brocket deer (Mazama americana and Mazama rufa) being the most impressive example. The chromosome variation was clustered in cytotypes with diploid numbers ranging from 42 to 53 and was correlated with geographical location. We conducted an analysis of chromosome evolution of the red brocket deer complex using comparative chromosome painting and Bacterial Artificial Chromosome (BAC) clones among different cytotypes. The aim was to deepen our understanding of the karyotypic relationships within the red brocket, thereby elucidating the significant chromosome variation among closely related species. This underscores the significance of chromosome changes as a key evolutionary process shaping their genomes. The results revealed the presence of three distinct cytogenetic lineages characterized by significant karyotypic divergence, suggesting the existence of efficient post-zygotic barriers. Tandem fusions constitute the main mechanism driving karyotype evolution, following a few centric fusions, inversion X-autosomal fusions. The BAC mapping has improved our comprehension of the karyotypic relationships within the red brocket deer complex, prompting questions regarding the role of these changes in the speciation process. We propose the red brocket as a model group to investigate how chromosomal changes contribute to isolation and explore the implications of these changes in taxonomy and conservation.

X Chromosome-Specific Repeats in Non-Domestic Bovidae.

Repetitive sequences form a substantial and still enigmatic part of the mammalian genome. We isolated repetitive DNA blocks of the X chromosomes of three species of the family Bovidae: Kobus defassa (KDEXr sequence), Bos taurus (BTAXr sequence) and Antilope cervicapra (ACEXr sequence). The copy numbers of the isolated sequences were assessed using qPCR, and their chromosomal localisations were analysed using FISH in ten bovid tribes and in outgroup species. Besides their localisation on the X chromosome, their presence was also revealed on the Y chromosome and autosomes in several species. The KDEXr sequence abundant in most Bovidae species also occurs in distant taxa (Perissodactyla and Carnivora) and seems to be evolutionarily older than BTAXr and ACEXr. The ACEXr sequence, visible only in several Antilopini species using FISH, is probably the youngest, and arised in an ancestor common to Bovidae and Cervidae. All three repetitive sequences analysed in this study are interspersed among gene-rich regions on the X chromosomes, apparently preventing the crossing-over in their close vicinity. This study demonstrates that repetitive sequences on the X chromosomes have undergone a fast evolution, and their variation among related species can be beneficial for evolutionary studies.

Impact of air pollution from different sources on sperm DNA methylation.

Environmental exposure is associated with increased incidence of respiratory and cardiovascular diseases and reduced fertility. Exposure to air pollution can influence gene expression through epigenetic mechanisms. In this study, we analysed gene-specific CpG methylation in spermatozoa of city policemen occupationally exposed to air pollution in two Czech cities differing by sources and composition of the air pollution. In Prague, the pollution is mainly formed by NO2 from heavy traffic. Ostrava is a hotspot of industrial air pollution with high concentrations of particular matter (PM) and benzo[a]pyrene (B[a]P). We performed genome-wide methylation sequencing using the SureSelectXT Human Methyl-Seq system (Agilent Technologies) and next-generation sequencing to reveal differentially methylated CpG sites and regions. We identified differential methylation in the region chr5:662169 - 663376 annotated to genes CEP72 and TPPP. The region was then analysed in sperm DNA from 117 policemen using targeted methylation sequencing, which proved its hypermethylation in sperm of Ostrava policemen.

Chromosome damage in regions with different levels of air pollution.

Air pollution is an important environmental factor influencing human health. In this study, we compared chromosome damage in city policemen from three cities in the Czech Republic: industrial Ostrava characterized by high levels of benzo[a]pyrene, Prague with heavy traffic emitting nitrogen oxides, and relatively clean Ceske Budejovice located in an area with predominantly agricultural activity. Chromosomal aberrations in lymphocytes were evaluated by fluorescence in situ hybridization with painting probes for chromosomes 1, 2, 3, and 4 in spring and autumn. An increase in the frequency of unstable chromosome aberrations, that is, dicentric chromosomes and acentric fragments, was observed in spring samples from Ostrava (p = .014 and p = .044, respectively) and Prague (p = .002 and p = .006, respectively) in comparison with Ceske Budejovice. The difference was significant only for samples taken after the winter period, when the concentration of pollutants in the air increases due to poor dispersion conditions. An increased frequency of dicentric chromosomes was observed in spring compared to autumn in both Ostrava and Prague (p = .017 and p = .023, respectively), but not in Ceske Budejovice. More breakpoints were observed on chromosome 1 than on the other chromosomes examined (p < .001). The number of breakpoints in the heterochromatin region 1p11-q12 was lower than in other parts of chromosome 1 (p < .001), suggesting a protective function of heterochromatin against damage. Our study showed, that air pollution increased the frequency of unstable chromosome aberrations, especially dicentric chromosomes. However, we did not show an effect on stable chromosome rearrangements.

Supernumerary Marker Chromosome Identified in Asian Elephant (Elephas maximus).

We identified a small, supernumerary marker chromosome (sSMC) in two phenotypically normal Asian elephants (Elephas maximus): a female (2n = 57,XX,+mar) and her male offspring (2n = 57,XY,+mar). sSMCs are defined as structurally abnormal chromosomes that cannot be identified by conventional banding analysis since they are usually small and often lack distinct banding patterns. Although current molecular techniques can reveal their origin, the mechanism of their formation is not yet fully understood. We determined the origin of the marker using a suite of conventional and molecular cytogenetic approaches that included (a) G- and C-banding, (b) AgNOR staining, (c) preparation of a DNA clone using laser microdissection of the marker chromosome, (d) FISH with commercially available human painting and telomeric probes, and (e) FISH with centromeric DNA derived from the centromeric regions of a marker-free Asian elephant. Moreover, we present new information on the location and number of NORs in Asian and savanna elephants. We show that the metacentric marker was composed of heterochromatin with NORs at the terminal ends, originating most likely from the heterochromatic region of chromosome 27. In this context, we discuss the possible mechanism of marker formation. We also discuss the similarities between sSMCs and B chromosomes and whether the marker chromosome presented here could evolve into a B chromosome in the future.

Chromosomal damage in occupationally exposed health professionals assessed by two cytogenetic methods.

The study assessed occupationally induced chromosomal damage in hospital personnel at risk of exposure to antineoplastic drugs and/or low doses of ionizing radiation by two cytogenetic methods. Cultured peripheral blood lymphocytes of eighty-five hospital workers were examined twice over 2 to 3 years by classical chromosomal aberration analysis and fluorescence in situ hybridization. The comparison of the 1st and the 2nd sampling of hospital workers showed a significant increase in chromatid and chromosomal aberrations (all p < .05) examined by classical chromosomal aberration analysis, and in unstable aberrations (all p < .05) detected by fluorescence in situ hybridization. Both cytogenetic methods were able to detect an increase of unstable aberrations in the 2nd sampling. The raised frequency of unstable cytogenetic parameters suggested higher recent exposure to genotoxic agents.

Spectrum of sperm mtDNA deletions in men exposed to industrial air pollution.

Sperm mtDNA status can serve as a molecular marker of oxidative stress and environmental exposure. High levels of air pollution may be associated with increased mitochondrial DNA (mtDNA) deletion rates in sperm. We compared the length spectra of sperm mtDNA deletions in semen samples collected from city policemen exposed to traffic and industrial air pollution in two seasons with different levels of air pollution. We used long-range PCR to amplify a fragment of mtDNA (8066 bp) frequently affected by deletions, visualized the PCR products by gel electrophoresis, and analysed aberrant bands corresponding to deleted mtDNA, using gel documentation software. The predominance of undeleted sperm mtDNA was accompanied by a variety of shorter PCR product lengths in the vast majority of sperm samples, in both seasons. Sperm mtDNA molecules and bands corresponding to long deletions were more frequently detected than shorter deletions, in both seasons. We did not detect any difference in the total number of electrophoretic bands corresponding to deleted sperm mtDNA and in the number of deleted sperm mtDNA molecules between the two seasons. In our study, air pollution during sperm maturation did not induce formation of large mtDNA deletions detectable by long PCR and gel electrophoresis (>1 kb) in maturing sperm mtDNA.

Cytogenetic Mapping of Cattle BAC Probes for the Hypothetical Ancestral Karyotype of the Family Cervidae.

Cervids are characterized by their greatest karyotypic diversity among mammals. A great diversity of chromosome numbers in notably similar morphological groups leads to the existence of several complexes of cryptic species and taxonomic uncertainties. Some deer lineages, such as those of Neotropical deer, stand out for a rapid chromosomal reorganization and intraspecific chromosome polymorphisms, which have not been properly explored yet. For that reason, we contribute to the study of deer karyotype diversity and taxonomy by producing and characterizing new molecular cytogenetic markers for the gray brocket deer (Subulo gouazoubira), a deer species that retained the hypothetical ancestral karyotype of Cervidae. We used bacterial artificial chromosome (BAC) clones derived from the cattle genome (Bos taurus) as markers, which were hybridized on S. gouazoubira metaphase chromosomes. In total, we mapped 108 markers, encompassing all gray brocket deer chromosomes, except the Y chromosome. The detailed analysis of fluorescent in situ hybridization results showed 6 fissions and 1 fusion as interchromosomal rearrangements that have separated cattle and gray brocket deer karyotypes. Each group of BAC probes derived from bovine chromosome pairs 1, 2, 5, 6, 8, and 9 showed hybridization signals on 2 different chromosomes, while pairs 28 and 26 are fused in tandem in a single acrocentric chromosome in S. gouazoubira. Furthermore, the BAC markers detected the occurrence of intrachromosomal rearrangements in the S. gouazoubira chromosomes homologous to pair 1 and the X chromosome of cattle. We present a karyotypic map of the 108 new markers, which will be of great importance for future karyotypic evolution studies in cervids and, consequently, help in their conservation and taxonomy resolution.

Association between sperm mitochondrial DNA copy number and deletion rate and industrial air pollution dynamics.

The effects of air pollution on men's reproductive health can be monitored by evaluating semen quality and sperm DNA damage. We used real-time PCR to analyse the effects of air pollution on sperm mitochondrial DNA copy number (mtDNAcn) and deletion (mtDNAdel) rates in semen samples collected from 54 men in two seasons with different levels of industrial and traffic air pollution. MtDNAdel rates were significantly higher following the high exposure period and were positively correlated with mtDNAcn. However, we did not find any difference in mtDNAcn between the two seasons. MtDNAcn was positively correlated with the DNA fragmentation index and the rates of sperm with chromatin condensation defects, previously assessed by sperm chromatin structure assay, and negatively correlated with sperm concentration, progressive motility, viability, and normal morphology. This indicates that mtDNAcn is more closely associated with male fertility than mtDNAdel rates. In contrast, mtDNAdel might be a more sensitive biomarker of air pollution exposure in urban industrial environments.

Effects of the air pollution dynamics on semen quality and sperm DNA methylation in men living in urban industrial agglomeration.

Human populations living in urban industrial regions of developed countries are exposed to high levels of environmental pollutants. The reproductive consequences of the exposure to air pollution can be monitored through semen analysis and molecular methods. In this study, we tested the possible impact of seasonal changes in the level of air pollution on the semen quality and sperm DNA methylation of 24 men living and working in the industrial agglomeration of Ostrava (Czech Republic). The study participants were healthy non-smokers. The study group was homogeneous regarding their profession, moderate alcohol consumption, no drug abuse and no additional exposure to chemical toxicants. We performed targeted methylation next generation sequencing (NGS) using Agilent SureSelect Human Methyl-Seq and Illumina NextSeq 500 platform to analyze semen samples collected repeatedly from the same men following the season of high (winter) and low (summer) air pollution exposure. We did not detect any adverse effects of the increased exposure on the semen quality; neither we found any difference in average sperm DNA methylation between the two sampling periods. Our search for differentially methylated CpG sites did not reveal any specific CpG methylation change. Our data indicate that the seasonal changes in the level of the air pollution probably do not have any substantial effect on sperm DNA methylation of men living in the highly polluted industrial agglomeration for a long period of time.

Genome-Wide DNA Methylation in Policemen Working in Cities Differing by Major Sources of Air Pollution.

DNA methylation is the most studied epigenetic mechanism that regulates gene expression, and it can serve as a useful biomarker of prior environmental exposure and future health outcomes. This study focused on DNA methylation profiles in a human cohort, comprising 125 nonsmoking city policemen (sampled twice), living and working in three localities (Prague, Ostrava and Ceske Budejovice) of the Czech Republic, who spent the majority of their working time outdoors. The main characterization of the localities, differing by major sources of air pollution, was defined by the stationary air pollution monitoring of PM2.5, B[a]P and NO2. DNA methylation was analyzed by a genome-wide microarray method. No season-specific DNA methylation pattern was discovered; however, we identified 13,643 differentially methylated CpG loci (DML) for a comparison between the Prague and Ostrava groups. The most significant DML was cg10123377 (log2FC = -1.92, p = 8.30 × 10-4) and loci annotated to RPTOR (total 20 CpG loci). We also found two hypomethylated loci annotated to the DNA repair gene XRCC5. Groups of DML annotated to the same gene were linked to diabetes mellitus (KCNQ1), respiratory diseases (PTPRN2), the dopaminergic system of the brain and neurodegenerative diseases (NR4A2). The most significant possibly affected pathway was Axon guidance, with 86 potentially deregulated genes near DML. The cluster of gene sets that could be affected by DNA methylation in the Ostrava groups mainly includes the neuronal functions and biological processes of cell junctions and adhesion assembly. The study demonstrates that the differences in the type of air pollution between localities can affect a unique change in DNA methylation profiles across the human genome.

Anchoring the CerEla1.0 Genome Assembly to Red Deer (Cervus elaphus) and Cattle (Bos taurus) Chromosomes and Specification of Evolutionary Chromosome Rearrangements in Cervidae.

The family Cervidae groups a range of species with an increasing economic significance. Their karyotypes share 35 evolutionary conserved chromosomal segments with cattle (Bos taurus). Recent publication of the annotated red deer (Cervus elaphus) whole genome assembly (CerEla1.0) has provided a basis for advanced genetic studies. In this study, we compared the red deer CerEla1.0 and bovine ARS-UCD1.2 genome assembly and used fluorescence in situ hybridization with bovine BAC probes to verify the homology between bovine and deer chromosomes, determined the centromere-telomere orientation of the CerEla1.0 C-scaffolds and specified positions of the cervid evolutionary chromosome breakpoints. In addition, we revealed several incongruences between the current deer and bovine genome assemblies that were shown to be caused by errors in the CerEla1.0 assembly. Finally, we verified the centromere-to-centromere orientation of evolutionarily fused chromosomes in seven additional deer species, giving a support to previous studies on their chromosome evolution.

Semen quality and sperm DNA integrity in city policemen exposed to polluted air in an urban industrial agglomeration.

BACKGROUND: We examined sperm quality in a cohort of city policemen in Ostrava at the end of a period with high concentrations of air pollutants (winter) and in the same cohort at the end of a relatively low exposure period (summer). METHODS: The study group was comprised of 54 nonsmoking city policemen living and working in Ostrava, Czech Republic. Average daily air-pollutant concentrations recorded by stationary monitoring for 90 days preceding the collection of semen samples were evaluated for different city districts of Ostrava. Standard semen parameters were assessed according to the guidelines of the World Health Organization (2010). The parameters were semen volume, sperm concentration, sperm morphology, sperm motility, acrosome reaction and sperm plasma membrane integrity. Sperm DNA damage was analysed by the sperm chromatin structure assay (SCSA). Sperm motion characteristics were determined by Computer Assisted Semen Analysis (CASA). RESULTS: The concentrations of all monitored pollutants (particulate matter, sulphur dioxide, nitrogen oxides, carbon monoxide, benzo[a]pyrene, benzene) were significantly increased during winter (p < 0.001), except for ozone, the concentration of which was significantly higher during summer. Sperm volume, concentration, % vitality, % sperm morphology (normal form) and % acrosome-intact sperm did not differ significantly between the monitoring periods. The percentages of total motility and progressive motility were significantly higher in March, i.e. at the end of winter (p = 0.001). However, CASA testing showed differences in sperm motion kinetics between spring and autumn samples. In the spring samples, we found a significantly lower % of straightness (p = 0.044) and the length of straight-line path (p = 0.01), while linearity and straight-line velocity were near the borderline value (p = 0.064; p = 0.054, respectively). As compared to summer, high exposure to air pollution during winter significantly increased the extent of sperm chromatin integrity damage (median 22.6 vs. 18.6%) (p = 0.003) and the proportion of immature spermatozoa (median 11.2 vs. 9.9%, p = 0.001). Sperm DNA damage negatively correlated with total motility and progressive motility (r = -0.611, -0.299; p < 0.001). The negative correlation with vitality, normal morphology and acrosome-intact sperm (r = -0.522, -0.550 and -0.511, respectively) was also significant (p < 0.001). CONCLUSION: The examination of the same cohort of city policemen at the end of a period of high air pollution and at the end of relatively low exposure reduced the effects of age, different lifestyles, different occupational exposures, localities and genetic polymorphism on sperm quality impairment associated with air pollution. This study did not demonstrate impaired standard semen parameters in association with exposure. It was shown that sperm chromatin damage and the percentage of immature sperm were highly sensitive to air pollution.

The effects of age on DNA fragmentation, the condensation of chromatin and conventional semen parameters in healthy nonsmoking men exposed to traffic air pollution.

BACKGROUND: Numerous studies have investigated age-based declines in semen traits, but the impact of paternal age on semen parameter values remains inconclusive. OBJECTIVES: The aim of this study was to detect an impact of age on semen quality was studied in healthy nonsmoking men exposed to traffic air pollution. METHODS: Semen samples from 150 Prague City policemen aged 23 to 63 years were examined for standard semen parameters, sperm DNA fragmentation and high DNA stainability. RESULTS: A significant positive correlation was found between age and %DFI (r = .359, P < .001), and negative correlations were found between age and sperm vitality (r = -.247, P < .001), the % acrosome-intact sperm (r = -.202, P = .013) and the % normal sperm heads (r = -.204, P = .012). A weak but significant negative correlation was found for high DNA stainability (% HDS) vs age (r = -.161, P = .050). No significant correlation was detected between male age and the other investigated semen quality parameters. At ages of 23 to 30, 31 to 40, 41 to 50, and 51 to 63 years, the mean %DFI values were 12.7 ± 7.18, 14.7 ± 7.42, 19.6 ± 11.25, and 34.2 ± 15.08, respectively. CONCLUSION: Our study shows a strong relationship (P < .001) between the age of men and sperm DNA fragmentation in an occupational cohort at risk of exposure to heavy traffic-related air pollution in a large city center.

Comparative chromosome painting in Chacoan peccary, Catagonus wagneri.

Chacoan peccary (Catagonus wagneri, 2n=20) is the most endangered of three extant species of Tayassuidae. Its karyotype has been studied only by differential chromosome staining methods so far. To establish a comparative cytogenetic map of the peccary, we used cross-species hybridization with porcine (Sus scrofa, 2n=38) painting probes. Painting revealed 30 evolutionary conserved autosomal segments between pig and peccary. The q-arm of the submetacentric chromosome X is homologous to the porcine X chromosome, while the p-arm is composed of heterochromatin. Nucleolar organizer regions were detected on chromosomes 8 and 9 which are homologous to pig chromosomes 8 and 4/18, respectively. Fusions of chromosomes homologous to pig chromosomes 4/7 and 4/18 and fission of chromosome 7 are synapomorphic characters shared by Catagonus wagneri and Tayassu pecari but not by Pecari tajacu. Our results confirmed a high rate of karyotype evolution in Tayassuidae and a closer relationship of Catagonus wagneri with Tayassu pecari than with Pecari tajacu.

Chromosomal Polymorphism and Speciation: The Case of the Genus Mazama (Cetartiodactyla; Cervidae).

Chromosomal polymorphism plays a major role in speciation processes in mammals with high rates of karyotypic evolution, as observed in the family Cervidae. One remarkable example is the genus Mazama that comprises wide inter- and intra-specific chromosomal variability. To evaluate the impact of chromosomal polymorphisms as reproductive barriers within the genus Mazama, inter-specific hybrids between Mazama gouazoubira and Mazama nemorivaga (MGO × MNE) and intra-specific hybrids between cytotypes of Mazama americana (MAM) differing by a tandem (TF) or centric fusion (Robertsonian translocations-RT) were evaluated. MGO × MNE hybrid fertility was evaluated by the seminal quality and testicular histology. MAM hybrids estimation of the meiotic segregation products was performed by sperm-FISH analysis. MGO × MNE hybrids analyses showed different degrees of fertility reduction, from severe subfertility to complete sterility. Regarding MAM, RT, and TF carriers showed a mean value for alternate segregation rate of 97.74%, and 67.23%, and adjacent segregation rate of 1.80%, and 29.07%, respectively. Our results suggested an efficient post-zygotic barrier represented by severe fertility reduction for MGO × MNE and MAM with heterozygous TF. Nevertheless, RT did not show a severe effect on the reproductive fitness in MAM. Our data support the validity of MGO and MNE as different species and reveals cryptic species within MAM.

Satellite DNA in Neotropical Deer Species.

The taxonomy and phylogenetics of Neotropical deer have been mostly based on morphological criteria and needs a critical revision on the basis of new molecular and cytogenetic markers. In this study, we used the variation in the sequence, copy number, and chromosome localization of satellite I-IV DNA to evaluate evolutionary relationships among eight Neotropical deer species. Using FISH with satI-IV probes derived from Mazama gouazoubira, we proved the presence of satellite DNA blocks in peri/centromeric regions of all analyzed deer. Satellite DNA was also detected in the interstitial chromosome regions of species of the genus Mazama with highly reduced chromosome numbers. In contrast to Blastocerus dichotomus, Ozotoceros bezoarticus, and Odocoileus virginianus, Mazama species showed high abundance of satIV DNA by FISH. The phylogenetic analysis of the satellite DNA showed close relationships between O. bezoarticus and B. dichotomus. Furthermore, the Neotropical and Nearctic populations of O. virginianus formed a single clade. However, the satellite DNA phylogeny did not allow resolving the relationships within the genus Mazama. The high abundance of the satellite DNA in centromeres probably contributes to the formation of chromosomal rearrangements, thus leading to a fast and ongoing speciation in this genus, which has not yet been reflected in the satellite DNA sequence diversification.

Sequence Analysis and FISH Mapping of Four Satellite DNA Families among Cervidae.

Centromeric and pericentromeric chromosome regions are occupied by satellite DNA. Satellite DNAs play essential roles in chromosome segregation, and, thanks to their extensive sequence variability, to some extent, they can also be used as phylogenetic markers. In this paper, we isolated and sequenced satellite DNA I-IV in 11 species of Cervidae. The obtained satellite DNA sequences and their chromosomal distribution were compared among the analysed representatives of cervid subfamilies Cervinae and Capreolinae. Only satI and satII sequences are probably present in all analysed species with high abundance. On the other hand, fluorescence in situ hybridisation (FISH) with satIII and satIV probes showed signals only in a part of the analysed species, indicating interspecies copy number variations. Several indices, including FISH patterns, the high guanine and cytosine (GC) content, and the presence of centromere protein B (CENP-B) binding motif, suggest that the satII DNA may represent the most important satellite DNA family that might be involved in the centromeric function in Cervidae. The absence or low intensity of satellite DNA FISH signals on biarmed chromosomes probably reflects the evolutionary reduction of heterochromatin following the formation of chromosome fusions. The phylogenetic trees constructed on the basis of the satellite I-IV DNA relationships generally support the present cervid taxonomy.

Recurrent gene mutations detected in canine mast cell tumours by next generation sequencing.

Genetic causes of canine mast cell tumours (MCTs), except for mutations in the KIT gene detected in some MCTs, are generally unknown. We used whole exome sequencing to reveal mutation spectra in canine MCTs. We detected somatic mutations in 87 genes including 10 genes recognized as human cancer drivers. Besides KIT, 14 other genes were recurrently mutated. Subsequently, we performed next generation sequencing of a panel of 50 selected genes in additional MCT samples. In this group, the most frequently altered gene was GNB1 showing a recurrent dinucleotide substitution at position of Gly116 in 30% of the MCT samples (n = 6/20) and Ile80 substitution accompanied by a splice region mutation in one case. We extended the study by analysis of the above mentioned GNB1 regions in additional MCT samples by Sanger sequencing, and assessed the overall prevalence of GNB1 mutations to 17.3% (n = 14/81), which is similar to the prevalence of KIT alterations. Our results indicate that GNB1 mutations are probably involved in canine MCT pathogenesis in both cutaneous and subcutaneous MCT cases. As opposed to KIT alterations, the presence of GNB1 mutations did not negatively affect survival times, and our data even showed a trend towards positive prognosis. If our results are confirmed in a larger number of MCTs, an extension of molecular testing of canine MCTs by GNB1 analysis would help to refine the molecular stratification of MCTs, and become useful for targeted treatment strategies.

Mutation and methylation status of KIT and TP53 in canine cutaneous and subcutaneous mast cell tumours.

Cutaneous and subcutaneous mast cell tumours (MCTs) are counted among the most frequent cancers in dogs. However, the genetic aetiology of their development is still mostly unknown, with the exception of KIT and tumor protein p53 (TP53 ) mutations reported in less than a half of cutaneous MCTs. In subcutaneous MCTs, no gene alterations were previously detected. We analysed KIT and TP53 mutations in cutaneous and subcutaneous MCTs, and identified methylated CpG sites in KIT and TP53 promoters and adjacent exon 1 regions. The mutation analysis focused on KIT exons 8, 9 and 11, and TP53 exons 5-8, and revealed mutations in 26% and 7% cutaneous MCT cases, respectively. Moreover, we report a first case of KIT mutation ever detected in subcutaneous MCTs. KIT exon 11 mutations and high Kiupel and Patnaik grades were associated with reduced survival in this study. Both KIT and TP53 gene were generally unmethylated in canine cutaneous MCTs. A sporadic methylation of the CpG positions in KIT promoter and adjacent exon 1 was detected in 70.4% of cutaneous and 82% of subcutaneous MCTs. A sporadic methylation of the CpG positions in the TP53 promoter and exon 1 was observed in 36.8% of the analysed cutaneous MCT samples. Only in two subcutaneous MCTs, we observed more than 30% of clones showing KIT methylation at the CpG positions 13 or 14. The CpG position 14 is involved in a predicted binding site for Sp1 transcription factor. However, the significance of KIT promoter methylation at this specific position needs further evaluation.