The use of molecular and cytogenetic methods as a valuable tool in the detection of chromosomal abnormalities in horses: a case of sex chromosome chimerism in a spanish purebred colt

Chromosomal abnormalities associated to sex chromo-somes are reported as a problem more common than be-lieved to be in horses. Most of them remain undiagnosed due to the complexity of the horse karyotype and the lack of interest of breeders and veterinarians in this type of diagno-sis. Approximately 10 years ago, the Spanish Purebred Breed-ers Association implemented a DNA paternity test to evalu-ate the pedigree of every newborn foal. All candidates who showed abnormal or uncertain results are routinely submit-ted to cytogenetical analysis to evaluate the presence of chromosomal abnormalities. We studied the case of a foal showing 3 and even 4 different alleles in several loci in the short tandem repeat (STR) -based DNA parentage test. To confirm these results, a filiation test was repeated using fol-licular hair DNA showing normal results. A complete set of conventional and molecular cytogenetic analysis was per-formed to determine their chromosomal complements. C-banding and FISH had shown that the foal presents a sex chimerism 64,XX/64,XY with a cellular percentage of approximately 70/30, diagnosed in blood samples. The use of a di-agnostic approach combining routine parentage QF-PCR-based STR screening tested with classical or molecular cyto-genetic analysis could be a powerful tool that allows early detection of foals that will have a poor or even no reproductive performance due to chromosomal abnormalities, saving time, efforts and breeders' resources

The use of a novel combination of diagnostic molecular and cytogenetical approaches in horses with sexual karyotype abnormalities: A rare case with an abnormal cellular chimerism

Sexual chromosomal aberrations are known to cause congenital abnormalities and unexplained infertility in horses. Most of these anomalies remain undiagnosed because of the complexity of the horse karyotype and the lack of specialized laboratories that can perform such diagnoses. On the other hand, the utilization of microsatellite markers is a technique widely spread in horse breeding, mostly due to their usage in parentage tests. We studied the usage of a novel combination of diagnostic approaches 25 in the evaluation of a very uncommon case of chromosomal abnormalities in a Spanish Purebred colt, primarily detected using a commercial panel of short tandem repeat (STR) makers. Based on these results we performed a full cytogenetical analysis using conventional and fluorescent in situ hybridization (FISH) techniques with individual ECAX (Equus caballus chromosome X) and ECAY (Equus caballus chromosome Y) painting probes. We also tested the presence of two genes associated with the sexual development in horses and an extra novel panel of 8 microsatellite markers specifically located in the sex chromosome pair. This is the first case reported of a leukocyte chimerism between a chromosomally normal (64,XY) and abnormal (63,X0) cell lines in horses. Our results indicate that the use of the STR markers as a screening technique and as a confirmation utilizing cytogenetic techniques can be used as a very interesting easy and non-expensive diagnostic approach to detect chromosomal abnormalities in the domestic horse

Sex reversal syndrome in the horse: Four new cases of feminization in individuals carrying a 64,XY SRY negative chromosomal complement

Horses are characterized as having a greater rate of chromosomal abnormalities than other species, which are mainly related to the sex chromosome pair and produce a series of different anomalies known as disorders in sexual development (DSD). In the present study, three Pura Raza Española (PRE) and one Menorquín (MEN) horses were studied and an incompatibility in their genetic and phenotypic sex were detected. Animals were karyotyped by conventional and molecular cytogenetic analyses and characterized using genomic techniques. Although all individuals, were totally unrelated, these animals had the same abnormality (64,XY SRY negative DSD) despite having an anatomically normal external mare phenotype. Therefore, this syndrome could remain undiagnosed in a large percentage of cases because the physiological and morphological symptoms are rare. In the present study, a slight gonadal dysgenesis was observed only in older individuals. Interestingly this chromosomal abnormality has been previously reported less than twenty times, and never in the PRE or MEN horses. With the present research, it is demonstrated that the use of genetic and cytogenetic diagnostic tools in veterinary practice could be an important complementary test to determine the origin of unexplained reproductive failures among horse

The Use of Genomic Screening for the Detection of Chromosomal Abnormalities in the Domestic Horse: Five New Cases of 65,XXY Syndrome in the Pura Raza Español Breed

Sex chromosomal abnormalities are a well-established cause of reproductive failure in domestic horses. Because of its difficult diagnosis, the Pura Raza Español breeding program established a routine screening for chromosomal abnormalities in all the horses prior to enrolling in the studbook. This genomic procedure combines an initial assessment based on the results from Short Tandem Repeat (STR) parentage testing followed by a Single-Nucleotide Polymorphism (SNP) based copy number aberration (CNA) confirmative analysis in positive cases. Using this methodology, we identified five new individuals carrying a 65,XXY chromosomal number aberration (CNA) among 27,330 foals enrolled over the past two reproductive seasons. The animals were initially flagged as CNA candidates due to abnormal results in STR testing. Subsequent analysis genotyping using an STR sex-linked dedicated panel and a medium-density SNP array in ECAX and ECAY confirmed the diagnosis as 65,XXY carriers. Four cases (upon sample availability) underwent further analysis using in situ fluorescent hybridization with ECAX and ECAY probes, showing identical results. Phenotypic analysis revealed abnormal gonad development in one of the cases, showing that the remaining four had a normal reproductive morphology. To our knowledge, this represents the largest number of horses exhibiting the equine form of Klinefelter syndrome (65,XXY) reported to date. Our study highlights the importance of genomic screening in the accurate detection of chromosomal abnormalities in horses

First case of sterility associated with sex chromosomal abnormalities in a jenny

Chromosomal abnormalities are one of the main causes of genetic infertility in horses. Currently, their detection rate is rising due to the use of new diagnostic tools employing molecular markers linked to the sex chromosome pair. Despite genetic similarities, there are no previous reports of sterility associated with chromosomal abnormalities in the domestic donkey (Equus asinus). Hereby, we determined the presence of a chromosomal mosaicism in a female donkey with reproductive problems using molecular methodologies developed for horses. A two-and- a- half- year- old jenny characterized by morphological abnormalities of the reproductive tract was cytogenetically analysed using conventional and fluorescent techniques and a group of microsatellite markers (short tandem repeat, STR). At the same time, five ultrasound measures of the reproductive tract were taken and compared with eight contemporary jennies of the same breed. After slaughter, morphological examinations showed that the case study had a blind vaginal vestibule defining an empty pouch that covered the entrance of the cervical os. Histopathological studies demonstrated that this abnormal structure was compatible with a remnant hymen. Molecular markers, STR and fluorescent in situ hybridization determinations revealed that the animal was a 62, XX/61,X mosaic and, therefore, the first case of chromosomal abnormalities in the sex pair reported in donkeys.Instituto de Genética Veterinari

First case of sterility associated with sex chromosomal abnormalities in a jenny

Chromosomal abnormalities are one of the main causes of genetic infertility in horses. Currently, their detection rate is rising due to the use of new diagnostic tools employing molecular markers linked to the sex chromosome pair. Despite genetic similarities, there are no previous reports of sterility associated with chromosomal abnormalities in the domestic donkey (Equus asinus). Hereby, we determined the presence of a chromosomal mosaicism in a female donkey with reproductive problems using molecular methodologies developed for horses. A two-and- a- half- year- old jenny characterized by morphological abnormalities of the reproductive tract was cytogenetically analysed using conventional and fluorescent techniques and a group of microsatellite markers (short tandem repeat, STR). At the same time, five ultrasound measures of the reproductive tract were taken and compared with eight contemporary jennies of the same breed. After slaughter, morphological examinations showed that the case study had a blind vaginal vestibule defining an empty pouch that covered the entrance of the cervical os. Histopathological studies demonstrated that this abnormal structure was compatible with a remnant hymen. Molecular markers, STR and fluorescent in situ hybridization determinations revealed that the animal was a 62, XX/61,X mosaic and, therefore, the first case of chromosomal abnormalities in the sex pair reported in donkeys.Instituto de Genética Veterinari

Low-basicity 5-HT7 receptor agonists synthesized using the van Leusen multicomponent protocol

A series of 5-aryl-1-alkylimidazole derivatives was synthesized using the van Leusen multicomponent reaction. The chemotype is the first example of low-basicity scaffolds exhibiting high affinity for 5-HT7 receptor together with agonist function. The chosen lead compounds 3-(1-ethyl-1H-imidazol-5-yl)-5- iodo-1H-indole (AGH-107, 1o, Ki 5-HT7=6nM, EC50=19nM, 176-fold selectivity over 5-HT1AR) and 1e (5-methoxy analogue, Ki 5-HT7=30nM, EC50=60nM) exhibited high selectivity over related CNS targets, high metabolic stability and low toxicity in HEK-293 and HepG2 cell cultures. A rapid absorption to the blood, high blood-brain barrier permeation and a very high peak concentration in the brain (Cmax=2723 ng/g) were found for 1o after i.p. (5mg/kg) administration in mice. The compound was found active in novel object recognition test in mice, at 0.5, 1 and 5mg/kg. Docking to 5-HT7R homology models indicated a plausible binding mode which explain the unusually high selectivity over the related CNS targets. Halogen bond formation between the most potent derivatives and the receptor is consistent with both the docking results and SAR. 5-Chlorine, bromine and iodine substitution resulted in a 13, 27 and 89-fold increase in binding affinities, respectively, and in enhanced 5-HT1AR selectivity

A Novel Mechanism of Tissue Inhibitor of Metalloproteinases-1 Activation by Interleukin-1 in Primary Human Astrocytes

Reactive astrogliosis is the gliotic response to brain injury with activated astrocytes and microglia being the major effector cells. These cells secrete inflammatory cytokines, proteinases, and proteinase inhibitors that influence extracellular matrix (ECM) remodeling. In astrocytes, the expression of tissue inhibitor of metalloproteinases-1 (TIMP-1) is up-regulated by interleukin-1 (IL-1), which is a major neuroinflammatory cytokine. We report that IL-1 activates TIMP-1 expression via both the IKK/NF-kappaB and MEK3/6/p38/ATF-2 pathways in astrocytes. The activation of the TIMP-1 gene can be blocked by using pharmacological inhibitors, including BAY11-7082 and SB202190, overexpression of the dominant-negative inhibitor of NF-kappaB (IkappaBalphaSR), or by the knock-down of p65 subunit of NF-kappaB. Binding of activated NF-kappaB (p50/p65 heterodimer) and ATF-2 (homodimer) to two novel regulatory elements located -2.7 and -2.2 kb upstream of the TIMP-1 transcription start site, respectively, is required for full IL-1-responsiveness. Mutational analysis of these regulatory elements and their weak activity when linked to the minimal tk promoter suggest that cooperative binding is required to activate transcription. In contrast to astrocytes, we observed that TIMP-1 is expressed at lower levels in gliomas and is not regulated by IL-1. We provide evidence that the lack of TIMP-1 activation in gliomas results from either dysfunctional IKK/NF-kappaB or MEK3/6/p38/ATF-2 activation by IL-1. In summary, we propose a novel mechanism of TIMP-1 regulation, which ensures an increased supply of the inhibitor after brain injury, and limits ECM degradation. This mechanism does not function in gliomas, and may in part explain the increased invasiveness of glioma cells

Design and synthesis of new quinazolin-4-one derivatives with negative mGlu7mGlu_7 receptor modulation activity and antipsychotic-like properties

Following the glutamatergic theory of schizophrenia and based on our previous study regarding the antipsychotic-like activity of mGlu7 NAMs, we synthesized a new compound library containing 103 members, which were examined for NAM mGlu7 activity in the T-REx 293 cell line expressing a recombinant human mGlu7 receptor. Out of the twenty-two scaffolds examined, active compounds were found only within the quinazolinone chemotype. 2-(2-Chlorophenyl)-6-(2,3-dimethoxyphenyl)-3-methylquinazolin-4(3H)-one (A9-7, ALX-171, mGlu7 IC50 = 6.14 µM) was selective over other group III mGlu receptors (mGlu4 and mGlu8), exhibited satisfactory drug-like properties in preliminary DMPK profiling, and was further tested in animal models of antipsychotic-like activity, assessing the positive, negative, and cognitive symptoms. ALX-171 reversed DOI-induced head twitches and MK-801-induced disruptions of social interactions or cognition in the novel object recognition test and spatial delayed alternation test. On the other hand, the efficacy of the compound was not observed in the MK-801-induced hyperactivity test or prepulse inhibition. In summary, the observed antipsychotic activity profile of ALX-171 justifies the further development of the group of quinazolin-4-one derivatives in the search for a new drug candidate for schizophrenia treatment

Genome Diversity and the Origin of the Arabian Horse

The Arabian horse, one of the world\u27s oldest breeds of any domesticated animal, is characterized by natural beauty, graceful movement, athletic endurance, and, as a result of its development in the arid Middle East, the ability to thrive in a hot, dry environment. Here we studied 378 Arabian horses from 12 countries using equine single nucleotide polymorphism (SNP) arrays and whole-genome re-sequencing to examine hypotheses about genomic diversity, population structure, and the relationship of the Arabian to other horse breeds. We identified a high degree of genetic variation and complex ancestry in Arabian horses from the Middle East region. Also, contrary to popular belief, we could detect no significant genomic contribution of the Arabian breed to the Thoroughbred racehorse, including Y chromosome ancestry. However, we found strong evidence for recent interbreeding of Thoroughbreds with Arabians used for flat-racing competitions. Genetic signatures suggestive of selective sweeps across the Arabian breed contain candidate genes for combating oxidative damage during exercise, and within the Straight Egyptian subgroup, for facial morphology. Overall, our data support an origin of the Arabian horse in the Middle East, no evidence for reduced global genetic diversity across the breed, and unique genetic adaptations for both physiology and conformation