A massive alteration of gene expression in undescended testicles of dogs and the association of KAT6A variants with cryptorchidism.

Cryptorchidism is the most common form of disorder of sex development in male dogs, but its hereditary predisposition is poorly elucidated. The gonadal transcriptome of nine unilaterally cryptorchid dogs and seven control dogs was analyzed using RNA-seq. Comparison between the scrotal and inguinal gonads of unilateral cryptorchid dogs revealed 8,028 differentially expressed genes (DEGs) (3,377 up-regulated and 4,651 down-regulated). A similar number of DEGs (7,619) was found by comparing the undescended testicles with the descended testicles of the control dogs. The methylation status of the selected DEGs was also analyzed, with three out of nine studied DEGs showing altered patterns. Bioinformatic analysis of the cDNA sequences revealed 20,366 SNP variants, six of which showed significant differences in allelic counts between cryptorchid and control dogs. Validation studies in larger cohorts of cryptorchid (n = 122) and control (n = 173) dogs showed that the TT genotype (rs850666472, p.Ala1230Val) and the AA genotype in 3'UTR (16:23716202G>A) in KATA6, responsible for acetylation of lysine 9 in histone H3, are associated with cryptorchidism (P = 0.0383). Both the transcript level of KAT6A and H3K9 acetylation were lower in undescended testes, and additionally, the acetylation depended on the genotypes in exon 17 and the 3'UTR. Our study showed that the massive alteration of the transcriptome in undescended testicles is not caused by germinal DNA variants in DEG regulatory sequences but is partly associated with an aberrant DNA methylation and H3K9 acetylation patterns. Moreover, variants of KAT6A can be considered markers associated with the risk of this disorder.

Altered Transcript Levels of MMP13 and VIT Genes in the Muscle and Connective Tissue of Pigs with Umbilical Hernia.

Umbilical hernia (UH) and inguinal hernia (IH) are among the most common defects in pigs, affecting their welfare and resulting in economic losses. In this study, we aimed to verify the association of previously reported differences in transcript levels of the ACAN, COL6A5, MMP13, and VIT genes with the occurrence of UH and IH. We examined mRNA levels in muscle and connective tissue from 68 animals-34 affected by UH and 34 controls. In a second cohort, we examined inguinal channel samples from 46 pigs (in four groups). We determined DNA methylation levels in muscle tissue for the UH and control animals. The transcript level of MMP13 changed in the UH cases, being upregulated and downregulated in muscle and connective tissue, respectively, and the VIT gene also showed an increased muscular mRNA level. The transcript of the ACAN gene significantly decreased in old pigs with IH. We further observed an increased DNA methylation level for one CpG site within the MMP13 gene in UH individuals. We conclude that these alterations in gene mRNA levels in the UH animals depend on the tissue and can sometimes be a consequence of, not a cause of, the affected phenotype.

Cleft Lip and Palate in Four Full-Sib Puppies from a Single Litter of Staffordshire Bull Terrier Dogs: An Anatomical and Genetic Study.

Cleft lip and palate (CLP) is a well-known congenital defect in dogs, characterized by abnormal communication between the oral and nasal cavities. Its incidence rate is high and affects all dog breeds. The etiology of CLP is thought to be multifactorial, caused by both genetic and environmental factors. In this study, four puppies out of seven from a single litter of Staffordshire Bull Terrier dogs with craniofacial abnormalities were anatomically and genetically examined. Classical anatomical preparation, dyed-latex-injection of the arterial vessels, and cone-beam computed tomography were used. The puppies showed variations in their observable abnormalities: three of them had a complete cleft of the palate on both sides, while one puppy had a cleft on the right side only. Cytogenetic analysis showed a normal diploid chromosome number (2n = 78,XX or 78,XY) in the studied animals. Known genomic variants of CLP were examined in the ADAMTS20, DLX6, and MYH3 genes, but no mutations were identified. Further studies are needed to identify the breed-specific genetic variants associated with canine CLP.

Non-mosaic X monosomy (77,X) in a female dog with signs of virilization.

A 14-month-old female Miniature Poodle dog with an enlarged clitoris and asymmetry in the placement of the teats was subjected to clinical, histopathological, and genetic studies. Macroscopically, the uterus and fallopian tubes appeared normal, while both ovaries were diffusely altered. At histology, the ovarian parenchyma was almost completely effaced by a diffuse hyperplasia of theca cells with atretic primary follicles. Chromosome analysis showed pure (non-mosaic) X monosomy (77,X). This finding was confirmed by the highly sensitive droplet digital PCR (ddPCR) approach. Despite the observed virilization, molecular analysis did not show the presence of Y-linked genes (SRY, ZFY, and TSPY1) in the blood cells or ovary tissue. To the best of our knowledge, this is the first case of X monosomy in a dog associated with virilization.

XX/XY Chimerism in Internal Genitalia of a Virilized Heifer.

Five DSD heifers underwent genetic analysis in the present study. We cytogenetically analyzed in vitro cultured leukocytes and searched for SRY, AMELX/AMELY and ZFX/ZFY genes in leukocytes and hair follicles, finding that four of the studied heifers were freemartins (XX/XY leukocyte chimerism). The fifth case had an underdeveloped vulva localized ventrally and cranially to the mammary gland, a normal female sex chromosome complement (60,XX) in the leukocytes, and a lack of Y-chromosome-derived genes in the leukocytes and hair follicles. Postmortem anatomical examination of this heifer revealed the presence of normal ovaries with follicles, uterus, and oviducts, but molecular detection of the SRY, ZFX, ZFY,AMELX, and AMELY genes in these organs indicated the presence of a cell line carrying the Y chromosome. Further analysis of twelve microsatellite markers revealed the presence of additional variants at six loci in DNA samples derived from the reproductive organs; XX/XY chimerism was thus suspected in these samples. On the basis of the detection of AMELY (Y-linked) versus AMELX (X-linked) and SOX9 (autosomal) versus AMELY genes by droplet digital PCR (ddPCR), the Y/X and Y/autosome ratios were evaluated; they indicated the presence of XX and XY cell lines in the reproductive tissues. Our study showed that XX/XY chimerism can be present in the internal reproductive organs of the virilized heifers with a normal female set of sex chromosomes (60,XX) and a lack of Y-chromosome-derived genes in the leukocytes. The etiology of this phenomenon remains unknown.

Cytogenetic and molecular insight into the genetic background of disorders of sex development in seventeen cats.

The genetic background of feline disorders of sex development (DSDs) is poorly understood. We performed comprehensive cytogenetic, molecular, and histological studies of 17 cats with abnormal external genitalia, unusual behavior, or tricolor coats (atypical in males). The DSD phenotype of three cats was associated with sex chromosome abnormalities: X/Y translocation (38,XXSRY+), 37,X/38,XY mosaicism, and XX/XY leukocyte chimerism. The remaining 14 affected cats were classified as XY DSD (SRY-positive). In this group and 38 normal males, we analyzed a priori selected candidate genes (SRY, TAC3, CYP11B1 and LHCGR). Only a previously reported nonpathogenic variant was found in SRY. Moreover, SRY gene copy number was determined, and three variants were observed: 6, 5 (modal), and 4 copies in a single DSD case. The known variants in TAC3 and CYP11B1, responsible for testicular hypoplasia, persistent primary dentition or congenital adrenal hyperplasia, were not found in the study group. Nine novel polymorphisms were identified in the LHCGR gene, one of which, a potentially regulatory indel variant in 5'UTR, was significantly associated (p = 0.0467) with XY DSD. Our report confirmed that abnormalities of sex chromosomes are important causes of feline DSDs. We also showed that the indel variant of LHCGR can be considered a promising marker associated with XY DSD phenotype.

Whole genome sequencing identifies a missense polymorphism in PADI6 associated with testicular/ovotesticular XX disorder of sex development in dogs.

Disorders of sex development (DSDs) are congenital malformations defined as discrepancies between sex chromosomes and phenotypical sex. Testicular or ovotesticular XX DSDs are frequently observed in female dogs, while monogenic XY DSDs are less frequent. Here, we applied whole genome sequencing (WGS) to search for causative mutations in XX DSD females in French Bulldogs (FB) and American Staffordshire Terries (AST) and in XY DSD Yorkshire Terries (YT). The WGS results were validated by Sanger sequencing and ddPCR. It was shown that a missense SNP of the PADI6 gene, is significantly associated with the XX DSD (SRY-negative) phenotype in AST (P = 0.0051) and FB (P = 0.0306). On the contrary, we did not find any associated variant with XY DSD in YTs. Our study suggests that the genetic background of the XX DSD may be more complex and breed-specific.

The Case of Atypical Sexual Attractiveness in a Male Domestic Dog-A Case Study.

During the ovarian cycle in domestic dogs, females do not accept males during the first days of estrus but become attractive to males from the beginning of proestrus, with this attractiveness persisting until the end of the estrus phase. It is believed that increased estradiol is responsible for the female attractiveness to the males. In this paper we describe the case of strong, but atypical attractiveness of a castrated male to various, adult, intact males, influenced by the emitted semiochemical signals. Any significant changes in the level of hormones typically involved in the process connected with estrus and responsible for sexual arousal in the males were assessed. The case animal was a 4 year old castrated male Border Collie that was extremely attractive to various males, which presented high levels of sexual arousal, with intensive sniffing and licking of the preputial area, specific vocalization, increased salivation and, finally, mating attempts. Clinical examination of the castrated male revealed a lack of testes in the scrotum and abdominal cavity confirmed by USG. Laboratory tests indicated basal levels of estradiol, testosterone, and progesterone (15.23 pg/mL, <0.05 ng/mL, 0.25 ng/mL), and sex was confirmed via cytogenetic and molecular analysis. Chemical analysis (HS-SPME) of the urine indicated a huge similarity to the profile obtained from a bitch in estrus, with an elevated level of acetophenone, which has been previously postulated in the literature as being a characteristic of the estrus phase in female domestic dogs. This case presented very atypical sexual attractiveness, particularly when taking into account the basal levels of hormones which, according to current knowledge, are responsible for the creation of attractiveness. As a hypothesis requiring verification, we propose the idea of involvement of other hormones in the creation of incidental attractiveness or increased production of compounds responsible for attractiveness (sex pheromones) resulting from metabolic events unrelated to reproductive processes. To our knowledge it is the first described case presenting this phenomenon, which, with more detailed study, could shed new light on the process of creation of sexual attraction in the domestic dog.

Identification of aneuploidy in dogs screened by a SNP microarray.

Microarray analysis is an efficient approach for screening and identifying cytogenetic imbalances in humans. SNP arrays, in particular, are a powerful way to identify copy-number gains and losses representing aneuploidy and aneusomy, but moreover, allow for the direct assessment of individual genotypes in known disease loci. Using these approaches, trisomies, monosomies, and mosaicism of whole chromosomes have been identified in human microarray studies. For canines, this approach is not widely used in clinical laboratory diagnostic practice. In our laboratory, we have implemented the use of a proprietary SNP array that represents approximately 650,000 loci across the domestic dog genome. During the validation of this microarray prior to clinical use, we identified three cases of aneuploidy after screening 2053 dogs of various breeds including monosomy X, trisomy X, and an apparent mosaic trisomy of canine chromosome 38 (CFA38). This study represents the first use of microarrays for copy-number evaluation to identify cytogenetic anomalies in canines. As microarray analysis becomes more routine in canine genetic testing, more cases of chromosome aneuploidy are likely to be uncovered.

Chromosome abnormalities in dogs with disorders of sex development (DSD).

Disorders of sex development (DSD) caused by chromosome abnormalities are rarely diagnosed in dogs. In this report, there is a focus on five DSD cases in which the dogs had abnormal karyotypes. All animals were recognized by owners as females, however, these dogs had a large number of reproductive defects. Among these were abnormal external genitalia such as an enlarged clitoris, abnormal development of the labia, abnormal location of the vulva and urethral orifice, and other abnormalities were observed in four dogs. Gonadal histology assessments were conducted on three dogs and there were diagnoses of the presence of an ovary, inactive testes, and ovotestis with calcification in ovarian follicles. Results from cytogenetic analysis indicated there were the following karyotypes: (a) X trisomy in a mosaic form (79,XXX/78,XX); (b) Robertsonian translocation in a mosaic form (77,XX,rob/78,XX); (c) nonmosaic X/autosome translocation (78,X,t(X;A)); (d) X/autosome translocation in a mosaic form (78,X,t(X;A)/78,XX); and (e) leukocyte chimerism (78,XX/78,XY). The findings in the present study, emphasize that cytogenetic analysis is essential for elucidating the pathogenesis of DSD in dogs.

Clinical Cytogenetics of the Dog: A Review.

The dog is an important companion animal and has been recognized as a model in biomedical research. Its karyotype is characterized by a high chromosome number (2n = 78) and by the presence of one-arm autosomes, which are mostly small in size. This makes the dog a difficult subject for cytogenetic studies. However, there are some chromosome abnormalities that can be easily identified, such as sex chromosome aneuploidies, XX/XY leukocyte chimerism, and centric fusions (Robertsonian translocations). Fluorescence in situ hybridization (FISH) with the use of whole-chromosome painting or locus-specific probes has improved our ability to identify and characterize chromosomal abnormalities, including reciprocal translocations. The evaluation of sex chromosome complement is an important diagnostic step in dogs with disorders of sex development (DSD). In such cases, FISH can detect the copy number variants (CNVs) associated with the DSD phenotype. Since cancers are frequently diagnosed in dogs, cytogenetic evaluation of tumors has also been undertaken and specific chromosome mutations for some cancers have been reported. However, the study of meiotic, gamete, and embryo chromosomes is not very advanced. Knowledge of canine genome organization and new molecular tools, such as aCGH (array comparative genome hybridization), SNP (single nucleotide polymorphism) microarray, and ddPCR (droplet digital PCR) allow the identification of chromosomal rearrangements. It is anticipated that the comprehensive use of chromosome banding, FISH, and molecular techniques will substantially improve the diagnosis of chromosome abnormalities in dogs.

A Disorder of Sex Development in a Holstein-Friesian Heifer with a Rare Mosaicism (60,XX/90,XXY): A Genetic, Anatomical, and Histological Study.

In this study, we describe an eighteen-month-old Holstein-Friesian heifer with a deformed vulva, located abdominally. The heifer showed typical signs of estrus. A comprehensive anatomical and histopathological examination revealed a blind-ended vagina and an additional section of urethra, which became a part of the shortened penis. Cytogenetic analysis showed the presence of two cell lines: 60,XX and 90,XXY. The frequency of the triploid cell line was low (3%) in leukocytes and elevated (35%) in fibroblasts. The molecular detection of Y-linked genes (SRY and AMELY) in the blood, skin, hair follicles, and buccal epithelial cells confirmed the presence of a cell line carrying the Y chromosome. Genotyping of 16 microsatellite markers in DNA isolated from hair follicles and fibroblast culture showed the presence of one (homozygous) or two variants (heterozygous) at all the studied loci, and allowed chimerism to be excluded. We concluded that the heifer had diploid/triploid (60,XX/90,XXY) mosaicism. To our knowledge, this is only the fifth such case to be reported worldwide in this species. Since cytogenetic studies are routinely performed on in vitro cultured leukocytes, we suspect that the prevalence of this chromosome abnormality is underestimated, as it is known from published reports that the frequency of the triploid cell line is usually very low in leukocytes.

Screening for structural variants of four candidate genes in dogs with disorders of sex development revealed the first case of a large deletion in NR5A1.

Disorders of sex development (DSD) are important causes of infertility and sterility, and are risk factors for gonadal carcinogenesis. Many DSDs are caused by genetic factors, mainly sex chromosome abnormalities or mutations of genes involved in sexual development, as well as structural variants (SVs) - large deletions, duplications, and insertions, if these overlap genes involved in sex development. The aim of this study was to determine if there were SVs in four candidate genes - NR0B1 (DAX1), NR5A1, RSPO1, and SOX3 - using droplet digital PCR (ddPCR). There was study of two cohorts of dogs with DSD, including 55 animals with XX DSD and 15 with XY DSD. In addition, 40 control females and 10 control males were included in the study. Among cases, for which there were evaluations, a large deletion consisting of four exons of the NR5A1 gene was identified in a Yorkshire Terrier with a rudimentary penis, hypospadias, bilateral cryptorchidism, and spermatogenesis inactive testes. This is the first mutation in the NR5A1 gene leading to XY DSD phenotype to be reported in domestic animals. There were no SVs in the genes evaluated in the present study in the cohort of dogs with XX DSD. The results from this study provide evidence that the large structural variants of these genes are rarely associated with the DSD phenotype in dogs.

Congenital Malformations in a Holstein-Fresian Calf with a Unique Mosaic Karyotype: A Case Report.

A Holstein-Fresian calf with multiple congenital malformations was subjected postmortem to anatomical and genetic investigation. The calf was small (20 kg), had shortened limbs and was unable to stand up. It lived only 44 days. Detailed anatomical investigation revealed the following features: head asymmetry, the relocation of the frontal sinus and eye orbits, hypoplastic thymus without neck part, ductus Botalli, unfinished obliteration in umbilical arteries, and a bilateral series of tooth germs in the temporal region. Cytogenetic examination, performed on in vitro cultured fibroblasts, showed a unique mosaic karyotype with a marker chromosome-60,XX[9 2%]/60,XX,+mar[8%], which was for the first time described in cattle. No other chromosome abnormalities indicating chromosome instabilities, like chromatid breaks or gaps were identified, thus teratogenic agent exposure during pregnancy was excluded. The marker chromosome (mar) was small and it was not possible to identify its origin, however, sequential DAPI/C (4',6-diamidino-2-phenylindole) band staining revealed a large block of constitutive heterochromatin, which is characteristic for centromeric regions of bovine autosomes. We suppose that the identified marker chromosome was a result of somatic deletion in an autosome and its presence could be responsible for the observed developmental malformations. In spite of the topographic distance among the affected organs, we expected a relationship between anatomical abnormalities. To the of our best knowledge, this is the first case of a mosaic karyotype with a cell line carrying a small marker chromosome described in a malformed calf.

Analysis of transcript and methylation levels of INSL3 and RXFP2 in undescended and descended dog testes suggested promising biomarkers associated with cryptorchidism.

Cryptorchidism is the most common disorder of sex development (DSD) in dogs. This malformation is associated with reduced fertility and with a higher risk of gonadal cancer. Testicular descent is a complex process, and the functions of many environmental and genetic factors are crucial for the proper migration of fetal gonads into the scrotum. Among these, the hormone INSL3 (insulin-like peptide 3) and its receptor RXFP2 (relaxin family peptide receptor 2) play crucial roles in the transabdominal migration of the testes. The genetic background of canine cryptorchidism is poorly elucidated. The aim of this study was to compare the transcript and methylation levels of INSL3 and RXFP2 genes in undescended and descended testes of isolated unilateral cryptorchids, and in gonads of control male dogs with scrotal testes. Next, we searched for polymorphic variants in the 5'-regulatory regions of both genes associated with predispositions to cryptorchidism. The INSL3 transcript level was significantly higher in the undescended testes than in the descended testes of both the affected and control dogs. On the other hand, the mRNA level of RXFP2 was significantly lower in the retained gonads of cryptorchids than in the scrotal testes. The methylation level of a single CpG site located 15 bp upstream of the translation start codon in INSL3 was significantly higher in the testes of the control dogs than in both gonads of cryptorchids. The methylation level of 14 CpG sites in the coding region of INSL3 was significantly higher in undescended testes than in the scrotal testes, which may be associated with the higher mRNA levels of INSL3 observed in these samples. The methylation pattern of two CpG sites in the 5'-flanking region of RXFP2 was similar in both descended and undescended testes. We detected three and seven single nucleotide polymorphisms (SNPs) in the 5'-regulatory regions of INSL3 and RXFP2, respectively. Among these, the frequency of A > C substitution (ss7093349755) located 495 bp upstream of the transcription start site of RXFP2 differed significantly between cryptorchids and control dogs. Our study showed two possible genetic biomarkers associated with canine cryptorchidism: a hypomethylation of a single CpG site in the 5'-flanking region of INSL3, and the ss7093349755 SNP in the 5'-flanking region of RXFP2.

Polymorphisms of CSF1R and WISP1 genes are associated with severity of familial adenomatous polyposis in APC1311 pigs.

Hereditary familial adenomatous polyposis (FAP) in humans significantly increases the risk of development of colorectal cancer (CRC). Germline mutations in the APC (adenomatous polyposis coli) gene are responsible for FAP. Despite having the same causative mutation, the severity of the disease differs from patient to patient. The porcine FAP model carrying a truncating APC1311 mutation, orthologous to the dominant human mutation that leads to severe form of the disease (APC1309), mirrors the severity of polyposis. Earlier RNAseq studies have revealed the differential expression of WISP1 and CSF1R in samples derived from low-grade (LG-IEN) and more advanced high-grade (HG-IEN) colon polyps of APC1311/+ pigs. The grade of dysplasia was correlated with the severity of polyposis in APC1311/+ pigs characterized by a low (LP) and high (HP) numbers of polyps. The goal of this work was to find DNA variants that regulate the expression of CSF1R and WISP1 in LP and HP pigs. In total, 32 and 36 polymorphisms in CSF1R and WISP1 were found, respectively. Of these, the genotype frequency of four silent SNPs in the coding region of WISP1 differed significantly between LP and HP lines. In silico analysis revealed an elevated minimum free energy (MFE) for three of these SNPs, suggesting their role in mRNA structure stability. Furthermore, four polymorphisms in the promoter region of CSF1R, cosegregating as a common haplotype, were associated with polyp number in APC1311/+ pigs. A secreted alkaline phosphatase (SEAP) assay showed, however, that these variants have no direct effect on the activity of the CSF1R promoter. Concluding, our study identified polymorphisms in CSF1R and WISP1 that are potentially associated with the severity of polyposis in APC1311/+ pigs.

Polymorphism of the growth hormone gene GH1 in Polish children and adolescents with short stature.

PURPOSE: Short stature in children is a significant medical problem which, without proper diagnosis and treatment, can lead to long-term consequences for physical and psychological health in adult life. Since human height is a polygenic and highly heritable trait, numerous variants in the genes involved in growth-including the growth hormone (GH1) gene-have been identified as causes of short stature. METHODS: In this study, we performed for the first time molecular analysis of the GH1 gene in a cohort (n = 186) of Polish children and adolescents with short stature, suffering from growth hormone deficiency (GHD) or idiopathic short stature (ISS), and a control cohort (n = 178). RESULTS: Thirteen SNP variants were identified, including four missense variants, six in 5'UTR, and three in introns. The frequency of minor missense variants was low (<0.02) and similar in the compared cohorts. However, two of these variants, Ala39Val (rs151263636) and Arg42Leu (rs371953554), were found (heterozygote status) in only two GHD patients. These substitutions, according to databases, can potentially be deleterious. CONCLUSIONS: Mutations of GH1 causing short stature are very rare in the Polish population, but two potentially causative variants need further studies in a larger cohort of GHD patients.