MicroRNA-146b negatively affects bovine embryo development and quality.

MicroRNAs (miRNAs), which can be carried inside extracellular vesicles (EVs), play a crucial role in regulating embryo development up to the blastocyst stage. Yet, the molecular mechanisms underlying blastocyst development and quality are largely unknown. Recently, our group identified 69 differentially expressed miRNAs in extracellular vesicles (EVs) isolated from culture medium conditioned by bovine embryos that either developed to the blastocyst stage or did not (non-blastocysts). We found miR-146b to be more abundant in the EVs derived from media conditioned by non-blastocyst embryos. Using RT-qPCR, we here confirmed the upregulation of miR-146b in non-blastocyst (arrested at 2-4 cell and morula stage) embryos compared to blastocysts (p<0.005), which coincides with the upregulation of miR-146b in EVs derived from the medium of these non-blastocysts. To evaluate a functional effect, bovine embryo culture media were supplemented with miR-146b mimics, resulting in significantly decreased embryo quality, with lower blastocyst rates at day 7 and lower total cell numbers, while the opposite was found after supplementation with miR-146b inhibitors, which resulted in reduced apoptosis rates (P < 0.01). Transcriptomic analysis of embryos treated with miR-146b mimics or inhibitors showed differential expression (P < 0.01) of genes associated with apoptosis, cell differentiation, and the RNA Pol II transcription complex, including WDR36, MBNL2, ERCC6l2, PYGO1, and SNIP1. Overall, miR-146b is overexpressed in non-blastocyst embryos and in EVs secreted by these embryos, and it regulates genes involved in embryo development and apoptosis, resulting in decreased embryo quality.

The TNNT2:c.95-108G>A variant is common in Maine Coons and shows no association with hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is a common and potentially fatal heart disease in many cat breeds. An intronic variant in TNNT2, c.95-108G>A, was recently reported as the cause of HCM in the Maine Coon. The aim of this study was to determine this variant's allele frequency in different populations and its possible association with HCM. Based on 160 Maine Coon samples collected in Belgium, Italy, Sweden and the USA, the variant's allele frequency was estimated to be 0.32. Analysis of the 99 Lives feline whole genome sequencing database showed that the TNNT2 variant also occurs in other breeds, as well as mixed-breed cats. Comparison of 31 affected and 58 healthy cats did not reveal significantly increased odds for HCM in homozygotes. Based on the combined evidence and in agreement with the standards and guidelines for the interpretation of sequence variants, this variant is currently classified as a variant of unknown significance and should not be used for breeding decisions regarding HCM.

Intrauterine growth restriction in neonatal piglets affects small intestinal mucosal permeability and mRNA expression of redox-sensitive genes.

Neonates with intrauterine growth restriction (IUGR) show lower efficiency of nutrient utilization compared to normal birth weight (NBW) newborns. This study was conducted using neonatal piglets as a model to test the hypothesis that IUGR affects the intestinal barrier function, intestinal structure, and antioxidant system development during the suckling period. The small intestinal mucosae were obtained from IUGR and NBW littermates in the suckling period (d 0, 3, 8, and 19 postnatal). The epithelial barrier function was assessed by FITC-dextran 4 (FD4) and horseradish peroxidase (HRP) fluxes across the epithelium, histomorphologic measurements, and expression of tight-junction proteins. Redox status represented by the glutathione disulfide/glutathione ratio and malondialdehyde concentrations was determined, whereas mRNA expressions of some redox-sensitive proteins were quantified. Results showed that IUGR piglets exhibited a 2-fold higher intestinal permeability in the proximal small intestine on d 0 (P < 0.05), and this difference between IUGR and NBW piglets was widened to 3 and 4 times for FD4 and HRP, respectively (P < 0.05), on d 3. In accordance, expression of occludin was down-regulated at the transcriptional level in IUGR piglets at d 0 and 19 (P < 0.01). Furthermore, the transcription of heme oxygenase 1, catalase, and thioredoxin reductase genes was down-regulated in IUGR piglets, mainly on postnatal d 0 and 19 (P < 0.01). It appears that IUGR subjects have a lower capacity to mount an antioxidant response in the early postnatal period. Collectively, these results add to our understanding of the mechanisms responsible for intestinal dysfunction in IUGR neonates.

High oxygen tension increases global methylation in bovine 4-cell embryos and blastocysts but does not affect general retrotransposon expression.

Retrotransposons are transposable elements that insert extra copies of themselves throughout the genome via an RNA intermediate using a 'copy and paste' mechanism. They account for more than 44% of the bovine genome and have been reported to be functional, especially during preimplantation embryo development. In the present study, we tested whether high oxygen tension (20% O2) influences global DNA methylation analysed by immunofluorescence staining of developing bovine embryos and whether this has an effect on the expression of some selected retrotransposon families. High oxygen tension significantly increased global DNA methylation in 4-cell embryos and blastocysts. A significant expression difference was observed for ERV1-1-I_BT in female blastocysts, but no significant changes were observed for the other retrotransposon families tested. Therefore, the study indicates that global DNA methylation is not necessarily correlated with retrotransposon expression in bovine preimplantation embryos.

Porcine EEF1A1 and EEF1A2 genes: genomic structure, polymorphism, mapping and expression.

Eukaryotic translation elongation factor 1 alpha (EEF1A) plays a key role in protein synthesis. In higher vertebrates EEF1A occurs in two isoforms, EEF1A1 and EEF1A2, encoded by distinct genes. The purpose of this study was to compare the two porcine genes as for the genomic sequence, gene organization and mRNA expression in different tissues, as well as to search for polymorphism and chromosomal assignment. Standard methods of DNA and mRNA analysis were used. We determined the complete genomic sequence of the porcine EEF1A1 and EEF1A2 genes. The two genes differ in the lengths of transcription units (3102 and 8588 bp, respectively), but have similar genomic organization and their coding sequences are highly similar (78% identity of coding sequences and 92.4% identity of amino acid sequences). Several polymorphisms in the two genes were detected. EEF1A1 and EEF1A2 were mapped to SSC1p11.1 and SSC17q23.3, respectively. mRNA of EEF1A1 was expressed in all studied tissues (the highest expression was in 44-day fetal muscle and low expression in adult liver and brain), while EEF1A2 was expressed only in skeletal-muscle, tongue, heart, diaphragm and brain tissues. EEF1A2 was not expressed in fetal muscle tissue (44 days). In this paper results are provided on genomic sequences, genomic organization, polymorphism, chromosomal assignment and spatial and temporal expressions of the porcine EEF1A1 and EEF1A2 genes. Novel polymorphisms were described in both genes. Porcine EEF1A2 was studied for the first time.

Variation in 12 porcine genes involved in the carbohydrate moiety assembly of glycosphingolipids does not account for differential binding of F4 Escherichia coli and their fimbriae.

BACKGROUND: Glycosphingolipids (GSLs) are important membrane components composed of a carbohydrate structure attached to a hydrophobic ceramide. They can serve as specific membrane receptors for microbes and microbial products, such as F4 Escherichia coli (F4 ETEC) and isolated F4 fimbriae. The aim of this study was to investigate the hypothesis that variation in genes involved in the assembly of the F4 binding carbohydrate moiety of GSLs (i.e. ARSA, B4GALT6, GAL3ST1, GALC, GBA, GLA, GLB1, GLB1L, NEU1, NEU2, UGCG, UGT8) could account for differential binding of F4 ETEC and their fimbriae. RESULTS: RT-PCR could not reveal any differential expression of the 12 genes in the jejunum of F4 receptor-positive (F4R(+)) and F4 receptor-negative (F4R(-)) pigs. Sequencing the complete open reading frame of the 11 expressed genes (NEU2 was not expressed) identified 72 mutations. Although some of them might have a structural effect, none of them could be associated with a F4R phenotype. CONCLUSION: We conclude that no regulatory or structural variation in any of the investigated genes is responsible for the genetic susceptibility of pigs towards F4 ETEC.

Expression of inflammation-related genes is associated with adipose tissue location in horses.

BACKGROUND: In humans, adipose tissue (AT) originating from different depots shows varying gene expression profiles. In horses, the risk of certain metabolic disorders may also be influenced by the impact of specific AT depots. Macrophage infiltration in human and rat AT is considered to be a source of inflammatory changes. In horses, this relationship has not been extensively studied yet. Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR), a useful method to evaluate differences in mRNA expression across different tissues, can be used to evaluate differences between equine AT depots. For a correct interpretation of the RT-qPCR results, expression data have to be normalized by the use of validated reference genes. The main objectives of this study were to compare mRNA expression of inflammation-related genes, as well as adipocyte morphology and number between different equine AT depots; and in addition, to investigate the presence of antigen presenting cells in equine AT and any potential relationship with adipokine mRNA expression. RESULTS: In this study, the mRNA expression of inflammation-related genes (leptin, chemokine ligand 5, interleukin 1ß, interleukin 6, interleukin 10, adiponectin, matrix metalloproteinase 2, and superoxide dismutase 2) and candidate reference gene stability was investigated in 8 different AT depots collected from the nuchal, abdominal (mesenteric, retroperitoneal, and peri-renal) and subcutaneous (tail head and loin) AT region. By using GeNorm analysis, HPRT1, RPL32, and GAPDH were found to be the most stable genes in equine AT. The mRNA expression of leptin, chemokine ligand 5, interleukin 10, interleukin 1ß, adiponectin, and matrix metalloproteinase 2 significantly differed across AT depots (P < 0.05). No significant AT depot effect was found for interleukin 6 and superoxide dismutase 2 (P > 0.05). Adipocyte area and number of antigen presenting cells per adipocyte significantly differed between AT depots (P < 0.05). CONCLUSIONS: Adipose tissue location was associated with differences in mRNA expression of inflammation-related genes. This depot-specific difference in mRNA expression suggests that the overall inflammatory status of horses could be partially determined by the relative proportion of the different AT depots.

Influence of the uterine environment on the development of in vitro-produced equine embryos.

The necessity for early interaction between the embryo and the oviductal and/or uterine environment in the horse is reflected by several striking differences between equine embryos that develop in vivo and those produced in vitro. Better understanding of the salient interactions may help to improve the efficiency of in vitro equine embryo production. In an initial experiment, cleavage-stage in vitro-produced (IVP) equine embryos were transferred into the uterus of recipient mares that had ovulated recently to determine whether premature placement in this in vivo environment would improve subsequent development. In a second experiment, an important element of the uterine environment was mimicked by adding uterocalin, a major component of the endometrial secretions during early pregnancy, to the culture medium. Intrauterine transfer of cleavage-stage IVP equine embryos yielded neither ultrasonographically detectable pregnancies nor day 7 blastocysts, indicating that the uterus is not a suitable environment for pre-compact morula stage horse embryos. By contrast, exposure to uterocalin during IVP improved capsule formation, although it did not measurably affect the development or expression of a panel of genes known to differ between in vivo and in vitro embryos. Further studies are required to evaluate whether uterocalin serves purely as a carrier protein or more directly promotes improved capsule development.

Gene expression profiling of pluripotency and differentiation-related markers in cat oocytes and preimplantation embryos.

During mammalian preimplantation development, two successive differentiation events lead to the establishment of three committed lineages with separate fates: the trophectoderm, the primitive endoderm and the pluripotent epiblast. In the mouse embryo, the molecular mechanisms underlying these two cell fate decisions have been studied extensively, leading to the identification of lineage-specific transcription factors. Species-specific differences in expression patterns of key regulatory genes have been reported, raising questions regarding their role in different species. The aim of the present study was to characterise the gene expression patterns of pluripotency (OCT4, SOX2, NANOG) and differentiation (CDX2, GATA6)-related markers during feline early development using reverse transcription-quantitative polymerase chain reaction. In addition, we assessed the impact of in vitro development on gene expression by comparing transcript levels of the genes investigated between in vitro and in vivo blastocysts. To normalise quantitative data within different preimplantation embryo stages, we first validated a set of stable reference genes. Transcript levels of all genes investigated were present and changed over the course of preimplantation development; a highly significant embryo-stage effect on gene expression was observed. Transcript levels of OCT4 were significantly reduced in in vitro blastocysts compared with their in vivo counterparts. None of the other genes investigated showed altered expression under in vitro conditions. The different gene expression patterns of OCT4, SOX2, CDX2 and GATA6 in cat embryos resembled those described in mouse embryos, indicative of a preserved role for these genes during early segregation. However, because of the absence of any upregulation of NANOG transcription levels after embryonic genome activation, it is unlikely that NANOG is a key regular of lineage segregation. Such results support the hypothesis that the behaviour of early lineage markers can be species specific. The present study also revealed a pool of maternal NANOG mRNA transcripts, the role of which remains to be elucidated. Comparing transcription levels of these genes between in vivo and in vitro blastocysts revealed low levels of OCT4 mRNA in the latter, which may contribute to the reduced developmental competence of embryos under suboptimal conditions.

Association of recessive c.430G>A (p.(Gly144Arg)) thyroid peroxidase variant with primary congenital hypothyroidism in cats.

BACKGROUND: Primary congenital hypothyroidism (CH) is a rare endocrine disorder in cats with a largely unknown genetic cause. OBJECTIVES: Describe the clinical presentation of CH in 11 affected cats and identify the causal genetic variant. ANIMALS: Eleven CH-cats from 10 unrelated families, 11 CH-free family members, 21 unrelated CH-free cats, and 155 unrelated nondiagnosed cats from different breeds. METHODS: Case control study of CH-cats and their siblings (2019-2021). Diagnosis was based on low to low-normal serum thyroxine (T4) concentrations, high thyroid-stimulating hormone (TSH) concentrations and clinical signs compatible with CH. We identified the causal variant using Sanger sequencing, genotyping via PCR-RFLP and variant interpretation using ACMG/AMP guidelines. RESULTS: All CH-cats (5 weeks-8 years) had disproportionate dwarfism. A goiter was not palpable in all. Thyroid scintigraphy with radiopertechnetate showed abnormally high uptake by thyroid glands, whereas scintigraphy with radioiodine showed abnormally low uptake, compatible with a defect in iodine organification by thyroid peroxidase (TPO). All cases were homozygous for TPO variant XM_006930524.4:c.430G>A(p.(Gly144Arg)), while none of the CH-free cats were. All sampled parents were heterozygous for this recessive variant. This variant was found in 15 cat breeds with an estimated allele frequency of 9%. CONCLUSIONS AND CLINICAL IMPORTANCE: Disproportionate dwarfism, abnormally high TSH and abnormally low to low-normal T4 concentrations are diagnostic for CH in cats. All cases had dyshormonogenesis demonstrated by thyroid scintigraphy. This novel TPO missense variant (not described in humans) causes CH in cats and awareness of it can assist in diagnosis and breeding.

Genetic Aspects of Corneal Sequestra in a Population of Persian, Himalayan and Exotic Cats.

Corneal sequestra are ophthalmic lesions that are remarkably common in Persian, Himalayan and exotic cats. In this study, the genetic aspects of this disease were investigated in a population of cats originating from a single cattery. Odds ratios were calculated for parents with affected offspring. The heritability of (owner-reported) corneal sequestra was estimated with a Markov chain Monte Carlo procedure. Well-phenotyped cases and controls were used for a genome-wide association study. Data from 692 cats originating from the cattery, of which 61 were affected, were used. Cats from two specific mothers had significantly higher odds of developing corneal sequestra, but no significant effect of the fathers was found (after correction for the mothers). The heritability of corneal sequestra was estimated to be 0.96. A genome-wide association study with 14 cases and 10 controls did not reveal an associated chromosomal region. The large effect that genetic factors had on the development of corneal sequestra in this study suggests that selective breeding could be an effective way to reduce the prevalence of this condition in these cat breeds.

Variation of inflammatory dynamics and mediators in primiparous cows after intramammary challenge with Escherichia coli.

The objective of the current study was to investigate (i) the outcome of experimentally induced Escherichia coli mastitis in primiparous cows during early lactation in relation with production of eicosanoids and inflammatory indicators, and (ii) the validity of thermography to evaluate temperature changes on udder skin surface after experimentally induced E. coli mastitis. Nine primiparous Holstein Friesian cows were inoculated 24 ± 6 days (d) after parturition in both left quarters with E. coli P4 serotype O32:H37. Blood and milk samples were collected before and after challenge with E. coli. The infrared images were taken from the caudal view of the udder following challenge with E. coli. No relationship was detected between severity of mastitis and changes of thromboxane B2 (TXB2), leukotriene B4 (LTB4) and lipoxin A4 (LXA4). However, prostaglandin E2 (PGE2) was related to systemic disease severity during E. coli mastitis. Moreover, reduced somatic cell count (SCC), fewer circulating basophils, increased concentration of tumor necrosis factor-α (TNF-α) and higher milk sodium and lower milk potassium concentrations were related to systemic disease severity. The thermal camera was capable of detecting 2-3 °C temperature changes on udder skin surface of cows inoculated with E. coli. Peak of udder skin temperature occurred after peak of rectal temperature and appearance of local signs of induced E. coli mastitis. Although infrared thermography was a successful method for detecting the changes in udder skin surface temperature following intramammary challenge with E. coli, it did not show to be a promising tool for early detection of mastitis.

In vivo-derived horse blastocysts show transcriptional upregulation of developmentally important genes compared with in vitro-produced horse blastocysts.

In vitro-produced (IVP) equine blastocysts can give rise to successful pregnancies, but their morphology and developmental rate differ from those of in vivo-derived equine blastocysts. The aim of the present study was to evaluate this difference at the genetic level. Suppression subtractive hybridisation (SSH) was used to construct a cDNA library enriched for transcripts preferentially expressed in in vivo-derived equine blastocysts compared with IVP blastocysts. Of the 62 different genes identified in this way, six genes involved in embryonic development (BEX2, FABP3, HSP90AA1, MOBKL3, MCM7 and ODC) were selected to confirm this differential expression by reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR). Using RT-qPCR, five genes were confirmed to be significantly upregulated in in vivo-derived blastocysts (i.e. FABP3, HSP90AA1 (both P<0.05), ODC, MOBKL3 and BEX2 (P<0.005 for all three)), confirming the results of the SSH. There was no significant difference in MCM7 expression between IVP and in vivo-derived blastocysts. In conclusion, five genes that are transcriptionally upregulated in in vivo-derived equine blastocysts compared with IVP blastocysts have been identified. Because of their possible importance in embryonic development, the expression of these genes can be used as a marker to evaluate in vitro embryo production systems in the horse.

Characterization of the ovine ribosomal protein SA gene and its pseudogenes.

BACKGROUND: The ribosomal protein SA (RPSA), previously named 37-kDa laminin receptor precursor/67-kDa laminin receptor (LRP/LR) is a multifunctional protein that plays a role in a number of pathological processes, such as cancer and prion diseases. In all investigated species, RPSA is a member of a multicopy gene family consisting of one full length functional gene and several pseudogenes. Therefore, for studies on RPSA related pathways/pathologies, it is important to characterize the whole family and to address the possible function of the other RPSA family members. The present work aims at deciphering the RPSA family in sheep. RESULTS: In addition to the full length functional ovine RPSA gene, 11 other members of this multicopy gene family, all processed pseudogenes, were identified. Comparison between the RPSA transcript and these pseudogenes shows a large variety in sequence identities ranging from 99% to 74%. Only one of the 11 pseudogenes, i.e. RPSAP7, shares the same open reading frame (ORF) of 295 amino acids with the RPSA gene, differing in only one amino acid. All members of the RPSA family were annotated by comparative mapping and fluorescence in situ hybridization (FISH) localization. Transcription was investigated in the cerebrum, cerebellum, spleen, muscle, lymph node, duodenum and blood, and transcripts were detected for 6 of the 11 pseudogenes in some of these tissues. CONCLUSIONS: In the present work we have characterized the ovine RPSA family. Our results have revealed the existence of 11 ovine RPSA pseudogenes and provide new data on their structure and sequence. Such information will facilitate molecular studies of the functional RPSA gene taking into account the existence of these pseudogenes in the design of experiments. It remains to be investigated if the transcribed members are functional as regulatory non-coding RNA or as functional proteins.

Suppression of keratin 18 gene expression in bovine blastocysts by RNA interference.

The expression of the cytoskeleton protein Keratin 18 (KRT18) starts at the onset of bovine blastocyst formation. KRT18 is solely expressed in the trophectoderm and can therefore be used as a marker for trophectodermal differentiation. In the present study, the expression of KRT18 was suppressed by RNA interference to probe its functional importance in bovine blastocyst formation. Microinjection of KRT18 double-stranded RNA into the cytoplasm of zygotes resulted in reduced KRT18 mRNA (76% reduction) and protein expression at the blastocyst stage and a lower developmental competence (41% reduction in the percentage of blastocyst formation) compared with non-injected and phosphate-buffered saline (PBS)-injected controls. KRT18 downregulation was associated with reduced mRNA expression of KRT8, the binding partner of KRT18, but had no effect on the expression of KRT19, CDH1 and DSP, other genes involved in intermediate filament and cytoskeleton formation. The results of the present study demonstrated that KRT18 knockdown in preimplantation embryos results in reduced blastocyst formation, but no further morphological aberrations were observed with regard to the biological function of KRT18. These observations could be due to the function of KRT18 being replaced by that of another gene, the surviving blastocysts expressing the minimum level of KRT18 required for normal blastocyst development or the possibility that further aberrations may occur later in development.

Quantification of fibronectin 1 (FN1) splice variants, including two novel ones, and analysis of integrins as candidate FN1 receptors in bovine preimplantation embryos.

BACKGROUND: Fibronectin 1 (FN1), a glycoprotein component of the extracellular matrix, exerts different functions during reproductive processes such as fertilisation, gastrulation and implantation. FN1 expression has been described to increase significantly from the morula towards the early blastocyst stage, suggesting that FN1 may also be involved in early blastocyst formation. By alternative splicing at 3 defined regions, different FN1 isoforms are generated, each with a unique biological function. The analysis of the alternative FN1 splicing on the one hand and the search for candidate FN1 receptors on the other hand during early bovine embryo development may reveal more about its function during bovine preimplantation embryo development. RESULTS: RT-qPCR quantification of the FN1 splice isoforms in oocytes, embryos, cumulus cells and adult tissue samples revealed a large variation in overall FN1 expression and in splice variant expression. Moreover, two new FN1 transcript variants were identified, the first one expressed in bovine preimplantation embryos and the second one expressed in cumulus cells. In the search for candidate receptors for the new embryo specific FN1 isoform, RNA expression analysis identified 5 alpha integrin subunits (ITGA2B, ITGA3, ITGA5, ITGA8, ITGAV) and 2 beta integrin subunits (ITGB1 and ITGB3) with a similar or overlapping RNA expression pattern as compared to FN1. But double immunofluorescent stainings could not confirm complete co-localisation between FN1 and one out of 3 selected integrins alpha subunits (ITGA3, ITGA5, ITGAV). CONCLUSION: The existence of a new FN1 transcript variant, specifically expressed in morulae and blastocysts strengthens the idea that FN1 is involved in the process of compaction and blastocyst formation. Analysis of the integrin expression could not identify the binding partner for the embryo specific FN1 transcript variant making further steps necessary for the identification of the FN1 receptor and the downstream effects of FN1-receptor binding.

Molecular cloning and characterization of the porcine prostaglandin transporter (SLCO2A1): evaluation of its role in F4 mediated neonatal diarrhoea.

BACKGROUND: Because prostaglandins are involved in many (patho)physiological processes, SLCO2A1 was already characterized in several species in an attempt to unravel specific processes/deficiencies. Here, we describe the molecular cloning and characterization of the porcine ortholog in order to evaluate its possible involvement in F4 enterotoxigenic E. coli mediated neonatal diarrhoea, based on a positional candidate gene approach study. RESULTS: Porcine SLCO2A1 is organized in 14 exons, containing an open reading frame of 1935 bp, encoding a 12-transmembrane organic anion cell surface transporter of 644 aa. The -388 to -5 upstream region comprises a (CpG)48 island containing a number of conserved promoter elements, including a TATA box. A potential alternative promoter region was found in the conserved -973 to -700 upstream region. No consensus polyadenylation signal was discovered in the 3' UTR. Repeat sequences were found in 15% of all the non coding sequences.As expected for a multifunctional protein, a wide tissue distribution was observed. mRNA expression was found in the adrenal gland, bladder, caecum, colon (centripetal coil/centrifugal coil), diaphragm, duodenum, gallbladder, heart, ileum, jejunum, kidney, liver, longissimus dorsi muscle, lung, lymph node, mesenterium, rectum, spleen, stomach, tongue and ureter, but not in the aorta, oesophagus and pancreas.The promoter region and the exons (including the splice sites) of SLCO2A1 were resequenced in 5 F4ab/ac receptor positive and 5 F4ab/ac receptor negative pigs. Two silent and 2 missense (both S --> L at position 360 and 633) mutations were found, but none was associated with the F4ab/ac receptor phenotype. In addition, no phenotype associated differential mRNA expression or alternative/abberant splicing/polyadenylation was found in the jejunum. CONCLUSION: The molecular cloning and characterization of porcine SLCO2A1 not only contributes to the already existing knowledge about the transporter in general, but enables studies on porcine prostaglandin related processes/deficiencies as patient and/or model. Here we examined its possible involvement as receptor in F4 enterotoxigenic E. coli mediated neonatal diarrhoea. Because no phenotype associated differences could be found in the gene sequence nor in its jejunal transcription profile of F4ab/ac receptor positive/negative pigs, SLCO2A1 can most likely be excluded as receptor for F4 bacteria.

Peripartal feeding strategy with different n-6:n-3 ratios in sows: effect on gene expression in backfat white adipose tissue postpartum.

The aim of this study was to describe the effects of two diets differing in n-6:n-3 ratio and prepartal feeding regime on gene expression of PPARgamma1a/1b, PPARgamma1c/1d, PPARgamma2, PPARgamma coactivator 1A (PPARGC1A), GLUT4, TNFalpha, adiponectin, leptin, leptin receptor (LEPR), fatty acid binding protein 4 (FABP4), lipoprotein lipase (LPL) in sows' white adipose tissue on the first day of lactation. The relationship between mRNA expression of these genes and circulating insulin, leptin and thyroid hormones was also considered. Diets contained a low (supplemented with fish oil; f group) or a high (supplemented with sunflower oil; s group) n-6:n-3 ratio and were provided from 8 (f8, s8) or 3d (f3, s3) before parturition (onset day 8 or 3). A low n-6:n-3 ratio reduced the 1d postpartum expression of PPARgamma2 and PPARGC1A but only when applied from 3 d before parturition. Circulating leptin was negatively correlated with mRNA expression of adiponectin, LEPR and LPL, whereas thyroxine was positively correlated with levels of PPARGC1A. In conclusion, the effect of dietary treatments, e.g. altering the n-6:n-3 ratio, around parturition on the expression of crucial genes in nutrient metabolism can be modulated by the duration of application before parturition.

A feline orthologue of the human MYH7 c.5647G>A (p.(Glu1883Lys)) variant causes hypertrophic cardiomyopathy in a Domestic Shorthair cat.

Hypertrophic cardiomyopathy (HCM) is the most common inherited human heart disease. The same disease has a high prevalence in cats, where it is also suspected to be inherited. More than 1500 variants in MYBPC3, MYH7 and other sarcomeric genes are associated with human HCM, while in cats, only two causative variants in MYBPC3 are currently known. Here, we describe an adult Domestic Shorthair cat with arterial thromboembolism and heart failure that was diagnosed with HCM on necropsy. Sequencing of the coding regions of MYBPC3 and MYH7 revealed 21 variants, of which the MYH7 c.5647G>A (p.(Glu1883Lys)) variant was further analysed, because its orthologous variant had already been reported in a human patient with HCM, but with limited causal evidence. This variant affects the highly conserved assembly competence domain, is predicted in silico to be damaging and was found only once in population databases. Recently, functional studies have confirmed its predicted damaging effect and a paralogous variant in MYH6 has been associated with cardiac disease in humans as well. This report of an orthologous variant in a cat with HCM and its absence in 200 additional cats provides further evidence for its disease-causing nature. As the first report of feline HCM caused by a variant in MYH7, this study also emphasises this gene as a candidate gene for future studies in cats and highlights the similarity between human and feline HCM.

Truncating SLC12A6 variants cause different clinical phenotypes in humans and dogs.

Clinical, pathological, and genetic findings of a primary hereditary ataxia found in a Malinois dog family are described and compared with its human counterpart. Based on the family history and the phenotype/genotype relationships already described in humans and dogs, a causal variant was expected to be found in KCNJ10. Rather surprisingly, whole-exome sequencing identified the SLC12A6 NC_006612.3(XM_014109414.2): c.178_181delinsCATCTCACTCAT (p.(Met60Hisfs*14)) truncating variant. This loss-of-function variant perfectly segregated within the affected Malinois family in an autosomal recessive way and was not found in 562 additional reference dogs from 18 different breeds, including Malinois. In humans, SLC12A6 variants cause "agenesis of the corpus callosum with peripheral neuropathy" (ACCPN, alias Andermann syndrome), owing to a dysfunction of this K+-Cl- cotransporter. However, depending on the variant (including truncating variants), different clinical features are observed within ACCPN. The variant in dogs encodes the shortest isoform described so far and its resultant phenotype is quite different from humans, as no signs of peripheral neuropathy, agenesis of the corpus callosum nor obvious mental retardation have been observed in dogs. On the other hand, progressive spinocerebellar ataxia, which is the most important feature of the canine phenotype, hindlimb paresis, and myokymia-like muscle contractions have not been described in humans with ACCPN so far. As this is the first report of a naturally occurring disease-causing SLC12A6 variant in a non-human species, the canine model will be highly valuable to better understand the complex molecular pathophysiology of SLC12A6-related neurological disorders and to evaluate novel treatment strategies.