An association and haplotype analysis of porcine maternal infanticide: a model for human puerperal psychosis?

An association analysis using the Illumina porcine SNP60 beadchip was performed to identify SNPs significantly associated with porcine maternal infanticide. We previously hypothesised that this was a good animal model for human puerperal psychosis, an extreme form of postnatal mood disorder. Animals were selected from carefully phenotyped unrelated infanticide and control groups (representing extremes of the phenotypic spectrum), from four different lines. Permutation and sliding window analyses and an analysis to see which haplotypes were in linkage disequilibrium (LD) were compared to identify concordant regions. Across all analyses, intervals on SSCs 1, 3, 4, 10, and 13 were constant, contained genes associated with psychiatric or neurological disorders and were significant in multiple lines. The strongest (near GWS) consistent candidate region across all analyses and all breeds was the one located on SSC3 with one peak at 23.4 Mb, syntenic to a candidate region for bipolar disorder and another at 31.9 Mb, syntenic to a candidate region for human puerperal psychosis (16p13). From the haplotype/LD analysis, two regions reached genome wide significance (GWS): the first on SSC4 (KHDRBS3 to FAM135B), which was significant (-logP 5.57) in one Duroc based breed and is syntenic to a region in humans associated with cognition and neurotism; the second on SSC15, which was significant (-log10P 5.68) in two breeds and contained PAX3, which is expressed in the brain.

The polymorphism analysis of CD169 and CD163 related with the risk of porcine reproductive and respiratory syndrome virus (PRRSV) infection.

Porcine reproductive and respiratory syndrome virus (PRRSV) could infect porcine alveolar macrophages (PAM), and the CD169 and CD163 are identified as critical receptors on the surface of PAM, but whether the single nucleotide polymorphisms (SNPs) of these genes could influence the infection is remain unclear. In this study, we identified totally 6 SNPs for CD169 (G1640T, C1654A, C4175T) and CD163 (G2277A, A2552G and C2700A), and evaluated their associations with PRRSV infection using two classified methods in a 524 pig population to investigate the effects of mutations on the PRRSV receptors. The pigs with genotypes of AA of CD169-C1654A, CT of CD169-C4175T and AA of CD163-A2552G appeared to resistant to the PRRSV infection by the combination of two classified results. The results provided fundamental molecular investigation to promote pig breeding with disease resistance. However, the identification of functional changes induced by SNPs and molecular mechanism were need further research.

Association of multi-pathogenic infections with BAT2, CXCL12, Mx1 and EHMT2 variations in pigs.

Porcine reproductive and respiratory syndrome virus (PRRSV), Haemophilus parasuis and Pseudorabies become a widespread problem causing great economic losses associated with reproductive disturbance, respiratory diseases, neonatal mortality, fibrinous polyserositis, meningitis and arthritis in the pig industry. The important candidate genes are assumed to play crucial roles in host defense against the diseases. The aims of this study were to evaluate the variants in HLA-B associated transcript 2 (BAT2), CXCL12, myxovirus resistance protein 1 (Mx1) and EHMT2 genes and their effects on the risk of infection PRRSV and H. parasuis in a case-control (diseased-healthy pigs) population of Duroc × Landrace × LargeWhite. The results showed that the mutations in BAT2, Mx1 and EHMT2 genes were significantly associated with the antibody and the reisk of infection PRRSV and H. parasuis. Those individuals with AA genotype of BAT2 had significantly higher Pseudorabies virus antibody than that with GG and GA (P < 0.05), and the individuals with TT genotype of EHMT2 generated higher Hog Cholera and Pseudorabies virus antibody than that wtih GG and GA (P < 0.01). These results indicated that the polymorphisms in Mx1, BAT2 and EHMT2 genes changed the diseases susceptibility and could be the potential markers assisting the pig breeding selection and disease resistance.

Effects of FUT1 gene mutation on resistance to infectious disease.

Alpha-(1,2)-fucosyltransferase (FUT1) gene has been identified as a candidate gene for regulating the expression of Escherichia coli F18 receptor gene (ECF18R) which promotes adherence of Enterotoxigenic (ETEC) and Verotoxigenic (VTEC) Escherichia coli (E. coli) via F18 fimbriae. In order to illustrate the polymorphisms of FUT1 and their effects on resistance to natural infection by Porcine Respiratory and Reproductive Symdrome Virus (PRRSV) and Haemophilus parasuis, the distributions of different genotypes and the relative risks of disease incidence in pigs were investigated. A total of 1,041 pigs representing three European breeds (Duroc, Landrace and LargeWhite), five Chinese local breeds (Wild pig, Small MeiShan, QinPing, JinHua, and JianLi) and three commercial populations (LargeWhite × JianLi, Duroc × Landrace × LargeWhite and Duroc × wild pig) were selected to analyze the genotype of the FUT1 gene by PCR-RFLP. Only the GG genotype associated with susceptibility to ECF18 bacteria was detected in Chinese local pig breeds and a population of LargeWhite × JianLi, while the AA genotype which confers resistance to ECF18 was detected in two European breeds (Duroc and LargeWhite), two populations of Duroc × wild pig and Duroc × Landrace × LargeWhite. Regarding relative risk of incidence, Duroc × Landrace × LargeWhite with genotypes GG or AG showed greater relative risk (OR = 2.040, P = 0.025; OR = 1.750, P = 0.081, respectively) than those with genotype AA during natural infection by both PRRSV and Haemophilus parasuis. It can be concluded that the mutation of FUT1 gene might play a role in pig infection by multi-pathogens, and that AA may be a favourable genotype for increasing the resistance to disease.

Effects of the polymorphisms of Mx1, BAT2 and CXCL12 genes on immunological traits in pigs.

It is necessary that genetic markers or biomarkers can be used to predict resistance towards a wide range of infectious diseases. In the present study, we estimated the potential markers and measured their relationship with heritabilities of a wide range of immune traits. Polymorphisms in exon 13 of Mx1, intron 25 of BAT2 and intron 3 of CXCL12 were identified by sequencing, and the genotypes were analyzed by PCR-RFLP in a resource population composed of 352 pure breed Landrace piglets at days 0, 17 and 32 after birth. Associations of single-nucleotide polymorphisms (SNPs) in these genes with a variety of immunological traits and antibody levels for pig reproduction and porcine respiratory syndrome virus (PRRSV), pseudorabies virus (PRV) and classical swine fever virus (CSFV) were performed. The performance of GG genotype of BAT2 on hemoglobin concentration (HBG) and hematocrit (HCT) of piglets at day 0 was significantly higher than that of the AA and AG individuals. For Mx1, compared with CT genotype, the pigs with TT or CC generated more PRRS antibody at day 0. The piglets with CT genotype had highly significant difference of PRV antibody from those with CC and TT genotypes at day 0. And the piglets with CC genotype had higher level red blood cell count (RBC), hemoglobin concentration (HBG) and hematocrit (HCT) than those with CT and TT genotypes at day 17. For the C7462G SNP in the intron 3 of CXCL12, the PRV antibody level of piglets with the CG genotype were higher than that of piglets with CC and GG genotypes at day 17, and the mean corpuscular volume (MCV) of GG piglets were larger than that of CC and CG individuals at day 0. At the locus 7331 bp in the intron 3 of CXCL12, there were significantly differences of mean corpuscular hemoglobin concentrations (MCHC) at day 0 and white blood cell count (WBC) at day 32, which showed the trend GG or AG>AA, AA>AG or GG, respectively. The pigs with AA or GG genotype had more platelet distribution width (PDW), mean platelet volume (MPV) and platelet-large cell ratio (PLR) at day 17 than those with AG. The results of this study indicated that polymorphisms in Mx1, BAT2 and CXCL12 genes were significantly associated with the immunological traits in Landrace piglets and had potential application value for marker-assisted selection of pig breeding with disease resistance.

Analysis of X chromosome genomic DNA sequence copy number variation associated with premature ovarian failure (POF).

BACKGROUND: Premature ovarian failure (POF) is a heterogeneous disease defined as amenorrhoea for >6 months before age 40, with an FSH serum level >40 mIU/ml (menopausal levels). While there is a strong genetic association with POF, familial studies have also indicated that idiopathic POF may also be genetically linked. Conventional cytogenetic analyses have identified regions of the X chromosome that are strongly associated with ovarian function, as well as several POF candidate genes. Cryptic chromosome abnormalities that have been missed might be detected by array comparative genomic hybridization. METHODS: In this study, samples from 42 idiopathic POF patients were subjected to a complete end-to-end X/Y chromosome tiling path array to achieve a detailed copy number variation (CNV) analysis of X chromosome involvement in POF. The arrays also contained a 1 Mb autosomal tiling path as a reference control. Quantitative PCR for selected genes contained within the CNVs was used to confirm the majority of the changes detected. The expression pattern of some of these genes in human tissue RNA was examined by reverse transcription (RT)-PCR. RESULTS: A number of CNVs were identified on both Xp and Xq, with several being shared among the POF cases. Some CNVs fall within known polymorphic CNV regions, and others span previously identified POF candidate regions and genes. CONCLUSIONS: The new data reported in this study reveal further discrete X chromosome intervals not previously associated with the disease and therefore implicate new clusters of candidate genes. Further studies will be required to elucidate their involvement in POF.

Transcriptional profiling of luteinizing hormone receptor-deficient mice before and after testosterone treatment provides insight into the hormonal control of postnatal testicular development and Leydig cell differentiation.

Luteinizing hormone (LH) is a key regulator of male fertility through its effects on testosterone secretion by Leydig cells. Transcriptional control of this is, however, currently poorly understood. Mice in which the LH receptor is knocked out (LuRKO) show reduced testicular size, reduced testosterone, elevated serum LH, and a spermatogenic arrest that can be rescued by the administration of testosterone. Using genome-wide transcription profiling of LuRKO and control testes during postnatal development and following testosterone treatment, we show that the transcriptional effects of LH insensitivity are biphasic, with an early testosterone-independent phase and a subsequent testosterone-dependent phase. Testosterone rescue re-enables the second, testosterone-dependent phase of the normal prepubertal transcription program and permits the continuation of spermatogenesis. Examination of the earliest responses to testosterone highlights six genes that respond rapidly in a dose-dependent fashion to the androgen and that are therefore candidate regulatory genes associated with the testosterone-driven progression of spermatogenesis. In addition, our transcriptional data suggest a model for the replacement of fetal-type Leydig cells by adult-type cells during testicular development in which a testosterone feedback switch is necessary for adult Leydig cell production. LH signaling affects the timing of the switch but is not a strict requirement for Leydig cell differentiation.

Global transcriptional response of pig brain and lung to natural infection by Pseudorabies virus.

BACKGROUND: Pseudorabies virus (PRV) is an alphaherpesviruses whose native host is pig. PRV infection mainly causes signs of central nervous system disorder in young pigs, and respiratory system diseases in the adult. RESULTS: In this report, we have analyzed native host (piglets) gene expression changes in response to acute pseudorabies virus infection of the brain and lung using a printed human oligonucleotide gene set from Illumina. A total of 210 and 1130 out of 23,000 transcript probes displayed differential expression respectively in the brain and lung in piglets after PRV infection (p-value < 0.01), with most genes displaying up-regulation. Biological process and pathways analysis showed that most of the up-regulated genes are involved in cell differentiation, neurodegenerative disorders, the nervous system and immune responses in the infected brain whereas apoptosis, cell cycle control, and the mTOR signaling pathway genes were prevalent in the infected lung. Additionally, a number of differentially expressed genes were found to map in or close to quantitative trait loci for resistance/susceptibility to pseudorabies virus in piglets. CONCLUSION: This is the first comprehensive analysis of the global transcriptional response of the native host to acute alphaherpesvirus infection. The differentially regulated genes reported here are likely to be of interest for the further study and understanding of host viral gene interactions.

Association analysis between pseudorabies antibody and five single-nucleotide polymorphisms in pigs.

Pseudorabies has become endemic and represents a widespread problem for pig production in the world, causing great economic losses associated with reproductive failure and neonatal mortality in the pig industry. Most diseases are the results of mutations of functional genes. Single-nucleotide polymorphisms (SNPs) from the coding regions of the mediators of pro-inflammatory responses or other candidate genes in pigs could indicate their potential involvement in susceptibility or resistance to PrV (pseudorabies virus) infection. There have been no previous association studies with candidate host genes that may influence PrV phenotypic traits. In order to perform association studies to identify genes contributing to PrV phenotypes, the genotypes of five SNPs from four genes (IL10, CXCL12, BAT2 and EHMT2) were determined for 178 sow samples using a high throughput microarray-based methodology. PrV antibodies were tested by enzyme-linked immunosorbent assay (ELISA) to determine whether there was an association between antibody levels and particular genotypes. The association between SNP genotypes and the PrV antibody levels were analysed using the Duncan method of one-way ANOVA procedure using the SAS (Statistical Analysis Systems) software package. The results showed that the glycoprotein E-ELISA antibody level of pigs with genotypes 11(AA) and 12(AG) was significantly higher than in pigs with genotype 22(GG) (P < 0.05) of SNP in the gene EHMT2-SNP2. The SNP of EHMT2 may be an effective potential tool to identify susceptible and resistant animals when used in conjunction with traditional selection methods.

Sex ratio bias in early-dead embryos of chickens collected during the first week of incubation.

According to Mendelian heredity laws, the sex ratio of a given chicken population during hatching is expected to be 1:1. In this study, we collected 432 chicken embryos that died during the first week of incubation from 5 different breeds. The sexes of the early-dead embryos were determined by using the previously described molecular sexing technique of double PCR. The female-to-male sex ratio was analyzed for departure from the expected 1:1 sex ratio by chi(2) testing. These results showed that the number of female dead embryos was significantly greater than that of males in the Hubei local breeding stock, Zhusi, and Hy-line Variety Brown (P < 0.05, P < 0.01, P < 0.01 respectively), with observed female-to-male sex ratios of 1.40:1, 2.03:1, and 2.22:1, respectively. Two other Chinese local breeds (the Yellow chicken and the Aijiaohuang chicken) also showed altered sex ratios, although the differences were not significant. Altogether, these results indicated that female chickens were more likely than male chickens to die at the early stages of incubation.

Identification of mutations of zona pellucida glycoprotein (ZP3) and its association with pig reproductive traits.

Reproduction is a complex trait, controlled by genetic and environmental factors. Genetic improvement of this trait is important for animal breeders to improve the animal's production efficiency. Apart from genetic factors, animal production can be affected by environmental factors, i.e. the nursing ability of the sow, which is in turn affected directly by effective teat number (teats producing milk normally, TN) and number of piglets born alive (NBA). The objective of this study was to find new mutations, such as single nucleotide polymorphisms (SNPs) from the Zona Pellucida glycoprotein gene (ZP3) using Single Strand Chain Polymorphism (SSCP) and nucleotide sequencing and to investigate association between genetic variations and sow reproductive traits. We identified 13 new SNPs from exon 1, two new SNPs from intron 2, one SNP from intron 6 and a 18 bp (GCACGTGGTCCTCCTGG)-deletion/insertion from intron 2 of the ZP3 gene. Five out of these mutations were selected to genotype in five different breeds (Small Meishan, Qingping, Duroc, Landrace and Large White) and association with reproductive traits in European breeds (Duroc, Landrace and Large White). The sows with genotype AA had more 1.11 piglets NBA than of the sows with genotype AB (p < 0.05) in the 18 bp deletion/insertion of intron 2, while non-significant associations between the other mutations and reproductive traits (NBA and TN) were found.

Analysis of sex chromosome abnormalities using X and Y chromosome DNA tiling path arrays.

BACKGROUND: Array comparative genomic hybridisation is a powerful tool for the detection of copy number changes in the genome. METHODS: A human X and Y chromosome tiling path array was developed for the analysis of sex chromosome aberrations. RESULTS: Normal X and Y chromosome profiles were established by analysis with DNA from normal fertile males and females. Detection of infertile males with known Y deletions confirmed the competence of the array to detect AZFa, AZFb and AZFc deletions and to distinguish between different AZFc lesions. Examples of terminal and interstitial deletions of Xp (previously characterised through cytogenetic and microsatellite analysis) have been assessed using the arrays, thus both confirming and refining the established deletion breakpoints. Breakpoints in iso-Yq, iso-Yp and X-Y translocation chromosomes and X-Y interchanges in XX males are also amenable to analysis. DISCUSSION: The resolution of the tiling path clone set used allows breakpoints to be placed within 100-200 kb, permitting more precise genotype/phenotype correlations. These data indicate that the combined X and Y tiling path arrays provide an effective tool for the investigation and diagnosis of sex chromosome copy number aberrations and rearrangements.

Association of four new single-nucleotide polymorphisms in follicle-stimulating hormone receptor and zona pellucida glycoprotein with reproductive traits in pigs.

Two new single-nucleotide polymorphisms (SNPs) (C1166T and G1190A) were discovered in the follicle-stimulating hormone receptor (FSHR) gene and two (G261A and T302C) in the zona pellucida glycoprotein (ZP3) gene. These SNPs were genotyped in three Chinese domestic purebred sow lines (42 Small Meishan, 46 Qingping and 41 Jinhua sows) and three European purebred sow lines (225 Duroc, 195 Large White and 65 Landrace sows) by using SNP chips. Phenotypic data including the functional teat number (i.e. milk-producing teats, TN) and number of piglets born alive per litter (NBA). These traits were tested for association with the genotypes of four SNPs. The association analysis revealed genotype of G261A in the ZP3 gene was significantly (P < 0.01) associated with overall NBA and NBA at later parities (NBA2+) but not with NBA at first parity (NBA1). There was a significant (P < 0.05) difference between sows with genotype GG (14.83 ± 0.18) and AA (14.26 ± 0.09) in TN at position 261 in the ZP3 gene. No significant associations were observed for the SNPs in the FSHR gene with NBA or TN in our populations. The results showed that the new SNPs in the ZP3 gene may be an effective potential marker to be used in conjunction with traditional selection methods.

Transcript profiles of some developmentally important genes detected in bovine oocytes and in vitro-produced blastocysts using RNA amplification and cDNA microarrays.

To study the mRNA transcript profiles of some potential candidate developmental genes during bovine oocyte and blastocyst stages, RNA amplification procedures, cDNA microarray of 82 target genes spotted onto glass slide and real-time polymerase chain reaction (PCR) were used. Messenger RNAs were isolated from in vitro-produced bovine matured oocytes and blastocysts. Using equal amounts of input mRNAs but different cycles of amplifications, cDNAs were produced and served as template for RNA amplification by the in vitro transcriptions. After amplification, the RNA yields transcribed from cDNAs of different cycles were evaluated both by hybridization on the cDNA microarrays and by using real-time PCR techniques. The analyses indicated best results from lower amplification cycle templates with consistent signals at hybridization. Generally, the RNA yield was directly proportional to the amplification cycle but inversely related with signal consistency at repeated hybridizations. Using the protocols established, equal amounts of amplified RNA from matured oocytes and blastocysts were hybridized to the array. Analyses of replicated hybridizations indicated that 35 transcripts were differentially expressed. Most of these were not described in previous bovine embryo studies. Independent analyses of 23 transcripts with real-time PCR and unamplified RNA confirmed the results of 22 genes. Moreover, the functional analyses showed various roles related to development. Hence, it is possible to conclude that the genes identified here are potential candidates for characterizing developmental competence, and that the methods established can be used for large-scale gene expression analysis with more comprehensive arrays.

Modulation of the mouse testis transcriptome during postnatal development and in selected models of male infertility.

The aim of this study is to develop an overview of genetic events during spermatogenesis using a novel, specifically targeted gonadal gene set. Two subtracted cDNA libraries enriched for testis specific and germ cell specific genes were constructed, characterized and sequenced. The combined libraries contain >1905 different genes, the vast majority previously uncharacterized in testis. cDNA microarray analysis of the first wave of murine spermatogenesis and of selected germ cell-deficient models was used to correlate the expression of groups of genes with the appearance of defined germ cell types, suggesting their cellular expression patterns within the testis. Real-time RT-PCR and comparison to previously known expression patterns confirmed the array-derived transcription profiles of 65 different genes, thus establishing high confidence in the profiles of the uncharacterized genes investigated in this study. A total of 1748 out of 1905 genes showed significant change during the first spermatogenic wave, demonstrating the successful targeting of the libraries to this process. These findings highlight unknown genes likely to be important in germ cell production, and demonstrate the utility of these libraries in further studies. Transcriptional analysis of well-characterized mouse models of infertility will allow us to address the causes and progression of the pathology in related human infertility phenotypes.

Breakpoint analysis of Turner patients with partial Xp deletions: implications for the lymphoedema gene location.

BACKGROUND: Turner syndrome is characterised by a 45,X karyotype and a variety of skeletal, lymphoedemic, and gonadal anomalies. Genes involved in the Turner phenotype are thought to be X/Y homologous with the X genes escaping X inactivation. Haploinsufficiency of the SHOX gene has been reported to cause the short stature seen in Turner syndrome patients. More recently, mutations of this gene have been shown to be associated with other skeletal abnormalities, suggesting that haploinsufficiency of SHOX causes all the Turner skeletal anomalies. No such gene has yet been identified for the lymphoedemic features. METHODS: Fluorescence in situ hybridisation (FISH) analysis with PAC clones on nine patients with partially deleted X chromosomes was performed. RESULTS/DISCUSSION: The Turner syndrome stigmata for each patient are described and correlation between the breakpoint and the phenotype discussed. A lymphoedema critical region in Xp11.4 is proposed and its gene content discussed with respect to that in the previously reported Yp11.2 lymphoedema critical region.

Insights into the molecular basis of male infertility.

The study of the genetic basis of human male infertility is complicated by genetic heterogeneity and because linkage analysis studies are difficult. The study has been limited so far to the analysis of genes located on the Y chromosome. Several genes and gene families have been discovered and mutation analysis of these candidate genes in infertile patients is ongoing. In recent years, several mouse models with impaired spermatogenesis or fertility have also been analysed, expanding our knowledge about the molecular basis of spermatogenesis and male fertility.

Characterization of the human Xq21.3/Yp11 homology block and conservation of organization in primates.

The Xq21.3/Yp11 homology block on the human sex chromosomes represents a recent addition to the Y chromosome through a transposition event. It is believed that this transfer of material occurred after the divergence of the hominid lineage from other great apes. In this paper we investigate the structure and evolution of the block through fluorescence in situ hybridisation, contig assembly, the polymerase chain reaction, exon trapping, sequence comparison, and annotation of sequence data. The overall structure is well conserved between the human X chromosome and the Y chromosome as well as between the X chromosomes from different primates. Although the sequence data reveal a high level of nucleotide sequence identity for the human X and Y, there are regions of significant divergence, such as that around the marker DXS214. These are presumably the consequence of multiple rearrangements during evolution and are of particular importance with respect to the potential gene content in this segment of the interval.