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Background Non-human primate (NHP) diabetic models using chemical ablation of β-cells with STZ have been achieved by several research groups. Chemotherapeutic STZ could lead to serious adverse events including nephrotoxicity, hepatotoxicity, and mortality. Methods We implemented a comprehensive therapeutic strategy that included the tether system, permanent indwelling catheter implants, an aggressive hydration protocol, management for pain with IV nubain and anxiety with IV midazolam, moment-by-moment monitoring of glucose levels post-STZ administration, and continuous intravenous insulin therapy. Results A triphasic response in blood glucose after STZ administration was fully characterized. A dangerous hypoglycemic phase was also detected in all baboons. Other significant findings were hyperglycemia associated with low levels of plasma leptin, insulin and C-peptide concentrations, hyperglucagonemia, and elevated non-esterified fatty acids (NEFA) concentrations. Conclusions We successfully induced frank diabetes by IV administering a single dose of pharmaceutical-grade STZ safely and without adverse events in conscious tethered baboons.

Cystinosis is an autosomal recessive disease characterised by the abnormal accumulation of lysosomal cystine. Mutations in the cystinosin gene (CTNS) represent known causes for the disease. The major cystinosis mutation is a 57 kb deletion on human chromosome 17p13 that removes the majority of CTNS and the entire adjacent gene, CARKL/SHPK.In order to identify other genes that may influence the cystinosis pathobiological pathway, peripheral blood mononuclear cells (PBMC) were collected from cystinosis family members, and DNA and RNA extracted.Using whole genome transcriptional profiling, transient receptor potential vanilloid 1 (TRPV1) was found to be differentially expressed in association with cystinosis. This was verified using TaqMan qRT-PCR. There was a 72% reduction in PBMC TRPV1 mRNA levels in cystinosis individuals homozygous for the 57 kb deletion (n=6) compared to unaffected individuals without the deletion (n=6) (p=0.002). TRPV1 is a sensory receptor located on chromosome 17p13, adjacent to CARKL/SHPK. It was ascertained that the 57 kb deletion extends from exon 10 of CTNS, upstream through CARKL/SHPK, to intron 2 of TRPV1, thus deleting the first two non-coding exons.This is the first study to report that the 57 kb deletion extends into the TRPV1 gene causing dysregulation of transcription in PBMC isolated from cystinosis patients.

Background Preeclampsia (PE) is a serious hypertensive pregnancy disorder with a significant genetic component. Numerous genetic studies, including our own, have yielded many susceptibility genes from distinct functional groups. Additionally, transcriptome profiling of tissues at the maternal-fetal interface has likewise yielded many differentially expressed genes. Often there is little overlap between these two approaches, although genes identified in both approaches are significantly associated with PE. We have thus taken a novel integrative bioinformatics approach of analysing pathways common to the susceptibility genes and the PE transcriptome. Methods Using Illumina Human Ht12v4 and Wg6v3 BeadChips, transcriptome profiling was conducted on n = 65 normotensive and n = 60 PE decidua basalis tissues collected at delivery. The R software package libraries lumi and limma were used to preprocess transcript data for pathway analysis. Pathways were analysed and constructed using Pathway Studio. We examined ten candidate genes, which are from these functional groups: activin/inhibin signalling—ACVR1, ACVR1C, ACVR2A, INHA, INHBB; structural components—COL4A1, COL4A2 and M1 family aminopeptidases—ERAP1, ERAP2 and LNPEP. Results/Conclusion Major common regulators/targets of these susceptibility genes identified were AGT, IFNG, IL6, INHBA, SERPINE1, TGFB1 and VEGFA. The top two categories of pathways associated with the susceptibility genes, which were significantly altered in the PE decidual transcriptome, were apoptosis and cell signaling (p < 0.001). Thus, susceptibility genes from distinct functional groups share similar downstream pathways through common regulators/targets, some of which are altered in PE. This study contributes to a better understanding of how susceptibility genes may interact in the development of PE. With this knowledge, more targeted functional analyses of PE susceptibility genes in these key pathways can be performed to examine their contributions to the pathogenesis and severity of PE.

Pre-eclampsia/eclampsia (PE/E) is a common, serious medical disorder of human pregnancy. Familial association of PE/E has been recognized for decades, but the genetics are complex and poorly understood. In an attempt to identify PE/E susceptibility genes, we embarked on a positional cloning strategy using 34 Australian and New Zealand PE/E pedigrees. An initial 10-cM resolution genome scan revealed a putative susceptibility locus spanning a broad region on chromosome 2 that overlaps an independently determined linkage signal seen in Icelandic PE pedigrees. Subsequent fine mapping using 25 additional short tandem repeat (STR) markers in this region and non-parametric multipoint linkage analysis did not change the overall position. Under a strict diagnosis of PE, we obtained significant evidence of linkage on 2q with a peak log-of-odds ratio score (LOD) of 3.43 near marker D2S151 at 155 cM. To prioritize positional candidate genes at the 2q locus for detailed analysis, we applied an objective prioritization strategy that integrates quantitative bioinformatics, assessment of differential gene expression and association analysis of single-nucleotide polymorphisms (SNPs). Highest priority was assigned to the activin receptor gene ACVR2. This gene also showed >10-fold differential gene expression in human decidual tissue from normotensive and PE individuals. We genotyped five known SNPs in this gene in our pedigrees and performed tests for association and linkage disequilibrium. One SNP (rs1424954) showed strong preliminary evidence of association with PE (P = 0.007), whereas two others (rs1364658 and rs1895694) exhibited nominal evidence (P < 0.05). Haplotype analysis revealed no additional association information. There was evidence of weak linkage disequilibrium among these SNPs. The highest observed LD occurred between the two strongest associated SNPs, suggesting that the observed signals may be the signature of an observed functional variant.

HLA-G is a nonclassical major histocompatibility complex class I (MHC-I) molecule that is primarily expressed at the fetal–maternal interface, where it is thought to play a role in protecting the fetus from the maternal immune response. HLA-G binds a limited repertoire of peptides and interacts with the inhibitory leukocyte Ig-like receptors LIR-1 and LIR-2 and possibly with certain natural killer cell receptors. To gain further insights into HLA-G function, we determined the 1.9-Å structure of a monomeric HLA-G complexed to a natural endogenous peptide ligand from histone H2A (RIIPRHLQL). An extensive network of contacts between the peptide and the antigen-binding cleft reveal a constrained mode of binding reminiscent of the nonclassical HLA-E molecule, thereby providing a structural basis for the limited peptide repertoire of HLA-G. The α3 domain of HLA-G, a candidate binding site for the LIR-1 and -2 inhibitory receptors, is structurally distinct from the α3 domains of classical MHC-I molecules, providing a rationale for the observed affinity differences for these ligands. The structural data suggest a head-to-tail mode of dimerization, mediated by an intermolecular disulfide bond, that is consistent with the observation of HLA-G dimers on the cell surface.

Epidemiological studies have shown that genetic factors contribute to the etiology of the common and serious pregnancy-specific disorder pre-eclampsia (PE)/eclampsia (E). Candidate-gene studies have provided evidence (albeit controversial) of linkage to several genes, including angiotensinogen on 1q42-43 and eNOS on 7q36. A recent medium-density genome scan in Icelandic families identified significant linkage to D2S286 (at 94.05 cM) on chromosome 2p12 and suggestive linkage to D2S321 (at 157.5 cM) on chromosome 2q23. In the present article, the authors report the results of a medium-density genome scan in 34 families, representing 121 affected women, from Australia and New Zealand. Multipoint nonparametric linkage analysis, using the GENEHUNTER-PLUS program, showed suggestive evidence of linkage to chromosome 2 (LOD=2.58), at 144.7 cM, between D2S112 and D2S151, and to chromosome 11q23-24, between D11S925 and D11S4151 (LOD=2.02 at 121.3 cM). Given the limited precision of estimates of the map location of disease-predisposing loci for complex traits, the present finding on chromosome 2 is consistent with the finding from the Icelandic study, and it may represent evidence of the same locus segregating in the population from Australia and New Zealand. The authors propose that the PE/E-linked locus on chromosome 2p should be designated the “PREG1” (pre-eclampsia, eclampsia gene 1) locus.

Objectives Preeclampsia (PE) is a common and serious pregnancy disorder with a strong genetic component. Numerous genetic studies performed, including our own, have yielded many susceptibility genes from distinct functional groups. Additionally, transcriptome profiling at the maternal-fetal interface has likewise yielded many differentially expressed genes. Often there is little overlap between the two approaches, although both are significantly informative with respect to PE. We have thus taken a novel approach by analysing pathways common to the susceptibility genes and the PE transcriptome. Methods Using Illumina Human Ht12v4 and Wg6v3 BeadChips, transcriptome profiling was conducted on n = 65 normotensive and n = 60 PE decidua basalis tissues collected at delivery. R software was used to pre-process transcript data for pathway analysis. Pathways were analysed and constructed using Ariadne Pathway Studio 9. We examined the following susceptibility genes: ACVR1, ACVR1C, ACVR2A, COL4A1, COL4A2, ERAP1, ERAP2, INHA, INHBB and LNPEP (Yong et al., 2014). Significance was set at p Results Major common targets/regulators of these susceptibility genes were AGT, IFNG, IL6, INHBA, SERPINE1, TGFB1 and VEGFA (Fig. 1). The top downstream pathway categories of susceptibility genes that were significantly altered in the PE decidual transcriptome were apoptosis and cell signalling. Conclusions Susceptibility genes from distinct functional groups have similar downstream pathways through common targets/regulators. Several of these pathways matched those altered in PE. This study contributes to a better understanding on how susceptibility genes may interact in the development of PE. With this knowledge, more targeted functional analyses of PE susceptibility genes in these key pathways can be performed to examine their contributions to the pathogenesis and severity of PE. Disclosures H.E. Yong: None. P.E. Melton: None. M.P. Johnson: None. K.A. Freed: None. B. Kalionis: None. P. Murthi: None. S.P. Brennecke: None. R.J. Keogh: None. E.K. Moses: None.

Preeclampsia (PE) is a serious complication of pregnancy, which is highly correlated with later life cardiovascular disease (CVD). Many risk factors are common for both diseases, but the contribution of shared genes remains to be determined. In this study, we used an integrative strategy to assess lipid traits as risk factors for PE and CVD by whole genome transcriptional profiling performed on Norwegian decidua basalis tissues (N = 95) from preeclamptic and normal pregnancies and on blood lymphocytes (N = 1240) from the San Antonio Family Heart Study (SAFHS). Among 222 genes that were differentially expressed (false discovery rate (FDR) P-value0.05) between the PE, cases and controls, we found one gene, ACOX2 (acyl-coenzyme A oxidase 2, branched chain), that was downregulated in PE whose transcription was also inversely correlated with triglyceride levels (P = 5.6 × 10(-7); FDR P-value = 0.0002) in SAFHS. We further report associations between SNPs in the ACOX2 gene and the transcription level (P-value = 0.0045) of the gene, as well as with triglyceride levels (P-value = 0.0051). ACOX2 is involved in bile acid production, a process that has been associated with both oxidative stress and regulation of triglyceride levels. Oxidative stress and increased triglyceride levels are known risk factors for CVD and both have also been associated with PE. Our results suggest that downregulation of ACOX2 is a shared risk factor for PE and CVD.

Differences in gene expression between cases and controls have been identified for a number of human diseases. However, the underlying mechanisms of transcriptional regulation remain largely unknown. Beyond comparisons of absolute or relative expression levels, disease states may be associated with alterations in the observed correlational patterns among sets of genes. Here we use partial correlation networks aiming to compare the transcriptional co-regulation for 222 genes that are differentially expressed in decidual tissues between preeclampsia (PE) cases and non-PE controls. Partial correlation coefficients (PCCs) have been calculated in cases (N = 37) and controls (N = 58) separately. For all PCCs, we tested if they were significant non-zero in the cases and controls separately. In addition, to examine if a given PCC is different between the cases and controls, we tested if the difference between two PCCs were significant non-zero. In the group with PE cases, only five PCCs were significant (FDR p value ≤ 0.05), of which none were significantly different from the PCCs in the controls. However, in the controls we identified a total of 56 statistically significant PCCs (FDR p value ≤ 0.05), of which 31 were also significantly different (FDR p value ≤ 0.05) from the PCCs in the PE cases. The identified partial correlation networks included genes that are potentially relevant for developing PE, including both known susceptibility genes (EGFL7, HES1) and novel candidate genes (CFH, NADSYN1, DBP, FIGLA). Our results might suggest that disturbed interactions, or higher order relationships between these genes play an important role in developing the disease.

Pre-eclampsia/eclampsia is a serious disorder of human pregnancy that has a worldwide incidence of 2-10% and carries a severe morbidity and mortality risk for both mother and child. Its precise cause remains unknown. However, there is increasing evidence of an underlying complex maternal genetic susceptibility. Its high population incidence in the face of strong negative selection pressure suggests that the gene(s) involved have a selective advantage and/or a high mutation rate. One class of genetic diseases that involve a high mutation rate are the trinucleotide repeat expansion diseases. Thus, the aim of this study was to determine whether there is an association between a trinucleotide (CAG) repeat expansion and pre-eclampsia/eclampsia. We have used the repeat expansion detection (RED) method, which was developed to directly identify clinically significant repeat expansions, to analyse genomic DNA from an Australian and New Zealand population. The maximal CAG repeat length for each individual was recorded and the Mann-Whitney U and Wilcoxon rank sum test for independent samples were used to compare distributions for CAG/CTG repeats between two populations. There were no statistically significant differences between the distribution of CAG repeats in normotensive (n = 59) and severe pre-eclampsia (n = 69) (Mann-Whitney U = 1732; P = 0.14), and normotensive (n = 59) and eclamptic (n = 15) populations (Mann-Whitney U = 417, P = 0.726). Therefore, these RED results do not support a role for a large CAG expansion in pre-eclampsia/eclampsia. However, these data do not preclude the possibility that a small CAG expansion is associated with the disorder nor do they negate the hypothesis that a highly mutable gene contributes to the genetic component of pre-eclampsia/eclampsia.

An aberrant interaction at the maternal/fetal interface between the genetically distinct fetal trophoblast cells and cells of the maternal decidua has been proposed as an initiating factor in one of the major complications of human pregnancy, preeclampsia. Biochemical and epidemiological studies suggest that the immune system plays an important role in preeclampsia. Thus, the aim of this study was to determine the decidual gene expression status in preeclampsia of one of the key components of the adaptive immune system. Total RNA was extracted from decidua collected from women with normal pregnancies and those complicated by preeclampsia. Reverse Northern analysis was performed on 72 cDNAs from human decidua and differentially expressed genes identified were analysed further using semi-quantitative RT-PCR and Northern blot analysis. Expression of the gene encoding the constant region of the heavy chain of immunoglobulin G (IgG CRHC) was shown to be down-regulated in association with preeclampsia. These data support the hypothesis that immune maladaptation may play an important role in the pathogenesis of preeclampsia.

Pre-eclampsia is a serious disorder of pregnancy that manifests clinically in the mother as new-onset hypertension and proteinuria. Although the precise cause remains unknown, the placenta and the decidua play a fundamental role. The worldwide incidence of pre-eclampsia is 2–5% and such a high incidence, in the face of strong negative selection, suggests that the gene(s) involved have a selective advantage and/or a high mutation rate. One class of genetic diseases that involve a high mutation rate are the trinucleotide repeat expansion diseases. In these diseases repeated trinucleotide DNA sequences within specific genes multiply or expand up to 1000-fold. The result of this gene expansion/mutation is altered gene function that confers genetic susceptibility. Thus, the overall objective of this study was to determine whether there is an association between a trinucleotide (CAG) repeat expansion and pre-eclampsia. The specific aim of this study was to isolate CAG repeat containing genes from human placenta and decidua. An adaptation of the mRNA differential display technique and traditional cDNA library screening was used. In total, 72 placental and 51 decidual sequences were analyzed using the BLAST nucleotide comparison program. Five cDNAs were analyzed further. The unique sequences surrounding the CAG repeat regions of these five genes will be used to generate primers to ascertain if any of these repeat DNA sequences vary in number in the normal population. If polymorphic genes are identified, the primers will be used on pre-eclamptic pedigrees to determine if pre-eclampsia is associated with a repeat expansion mutation.

Rabbit liver, a rich source of specific growth hormone (GH) receptors, contains three mRNA transcripts (4.2-4.5 kb, 3.1-3.2 kb, and 1.8-2.0 kb) which hybridize strongly to oligonucleotide probes complementary to nucleotide sequences in the extracellular and cytoplasmic regions of the rabbit liver GH receptor. The approximately 4.5 kb transcript was the most abundant and showed some sex difference (male greater than female) and a significant, approximately 2 fold increase in late pregnancy - observations consistent with changes seen in the specific 125I-hGH binding capacity of rabbit liver membranes prepared from the same tissue samples. The approximately 4.5 kb mRNA species, but not the smaller transcripts, was also detected, at lower abundance, in rabbit kidney, heart and lung but not in mammary gland, which is known to lack 125I-GH binding activity. These studies have identified the nature of the mRNA transcripts coding for the GH receptor in recognized/potential GH target tissues in the rabbit. The regulation of the major GH receptor mRNA in rabbit liver appears to broadly reflect known changes in expressed receptor protein.